Novel lanthionine antibiotic compositions and methods

ABSTRACT

According to the present invention, an isolated and purified DNA sequence which encodes a lantibiotic, mutacin I, is disclosed. The nucleic acid sequence is set forth in SEQ ID No: 1 and the amino acid sequence is set forth in SEQ ID No: 2.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 10/047,676 filed Jan. 14, 2002, now U.S. Pat. No. 6,699,970.application Ser. No. 10/047,676 is a divisional of U.S. patentapplication Ser. No. 09/627,376 filed Jul. 28, 2000, now U.S. Pat. No.6,342,285.

GRANT REFERENCE

[0002] The subject invention was made with government support under agrant from the National Institutes of Health (NIH RO 1 DE09082). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates to polypeptide antibiotics and to theidentification of genetic loci associated with expression of theantibiotics. The invention particularly describes a purifiedlanthionine-containing antimicrobial agent, DNA encoding the protein,and methods and compositions for treatments employing the antibiotic.

BACKGROUND OF THE INVENTION

[0004] Several species of bacteria inhabit the human oral cavity; amongthem Streptococcus mutans is considered a major etiologic agentresponsible for dental caries. Loesche (1986) Microbiol. Rev.50:353-380. Previous studies showed a certain percentage of clinicalisolates of S. mutans producing antimicrobial substances calledmutacins. Caufield et al. (1985) Infect. Immun. 48:51-56; Hamada et al.(1975) Arch. Oral Biol. 20:641-648. Mutacins are active against closelyrelated species as well as a surprisingly wide spectrum of otherGram-positive bacteria. Parrot et al. (1990) Can. J Microbiol.36:123-130. The ability to produce mutacins, combined with lactic acidproduction by S. mutans may contribute to the pathogenesis of thesebacteria. Kleinberg, p. 605-624, in W. A. Nolte (ed.), Oralmicrobiology, The C.V. Mosby Company, St. Louis. Production of mutacinsby S. mutans and other oral streptococci may also play a protective roleto the host against pathogens such as Group A streptococci andStreptococcus pneumoniae. In this respect, mutacins may serve asantimicrobial agents in the future.

[0005] Lantibiotics are lanthionine-containing small peptide antibioticsthat are produced by gram-positive bacteria. Jung (ed.), p. 1-34, in G.Jung and H. G. Sahl (ed.), Nisin and novel lantibiotics, ESCOM Sci.Publ., Leiden; Sahl et al. (1995) Eur. J Biochem. 230:827-853. Thelantibiotics are ribosomally synthesized and post-translationallymodified. The modification reactions include dehydration of serine andthreonine residues and the addition of thiol groups from cycteineresidues to the double bound to form lanthionines andβ-methyllanthionines, respectively. Some dehydrated serine or threoninemay remain as such in the mature lantibiotic molecule.

[0006] Based on the secondary structures, Jung assigned lantibioticsinto two classes, Type-A (linear) and Type-B (globular). Jung (ed.), p.1-34, in G. Jung and H. G. Sahl (ed.), Nisin and novel lantibiotics,ESCOM Sci. Publ., Leiden. de Vos et al. ((1995) Molecular Microbiol.17:427-437) and Sahl and Bierbaum (Sahl et al. (1998) Annu. Rev.Microbiol. 52:41-79) further divided each class into subgroups accordingto their primary peptide sequences. Thus, subgroup AI contains thenisin-like lantibiotics with nisin, subtilin, epidermin and pep5 as themost thoroughly characterized members. Allgaier et al. (1986) Eur. J.Biochem. 160:9-22; Gross et al. (1968) FEBS Lett. 2:61-64; Gross et al.(1971) J. Am. Chem. Soc. 93:4634-4635; Kaletta et al. (1989) Arch.Microbiol. 152:16-19; Weil et al. (1990) Eur. J Biochem. 194:217-223.Subgroup AII consists of lacticin 481, SA-FF22, salivaricin andvariacin. Hynes et al. (1993) Appl. Environ. Microbiol. 59:1969-1971;Piard et al. (1993) J. Biol. Chem. 268:16361-16368; Pridmore et al.(1996) Appl. Environ. Microbiol. 62:1799-1802; Ross et al. (1993) Appl.Environ. Microbiol. 59:2014-2021. The genes responsible for thebiosynthesis of the lantibiotics are organized in operon-likestructures. The biosynthesis locus of all members in the subgroup AIlantibiotics consists of lanA, the structural gene for the lantibiotic;lanB and lanC, the modifying enzyme genes for post-translationalmodification of the preprolantibiotic; lanP, the protease gene forprocessing of the prelantibiotic; and lanT, the ABC transporter forsecretion of the lantibiotic. In addition, epidermin and galliderminhave an extra gene, lanD, which is responsible for the C-terminaloxidative decarboxylation of the lantibiotic. Kupke et al. (1994) J.Biol. Chem. 269:5653-5659; Kupke et al. (1995) J. Biol. Chem.270:11282-89. In comparison, subgroup AII lantibiotics have simplergenomic organizations. In subgroup AII, lanB and lanC are combined intoone gene, lanM and lanP and lanT are combined into lanT. Chen et al.(1999) Appl. Environ. Microbiol. 65:1356-1360; Qi et al. (1999) Appl.Environ. Microbiol. 65:652-658; Rince et al. (1994) Appl. Environ.Microbiol. 60:1652-1657. All lantibiotic loci also contain a set ofimmunity genes, which are responsible for self-protection of theproducer strains. Saris et al. (1996) Antonie van Leewenhoek 69:151-159.Moreover, the expression of the lantibiotic genes is usually regulatedeither by a single transcription regulator (Peschel et al. (1993) Mol.Microbiol. 9:31-39; Qi et al. (1999) Appl. Environ. Microbiol.65:652-658) or by a two-component signal transduction system (de Ruyteret al. (1996) J. Bacteriol. 178:3434-3439; Klein et al. (1993) Appl.Environ. Microbiol. 59:296-303; Kuipers et al. (1995) J. Biol. Chem.270:27295-27304).

[0007] Previously, the isolation, biochemical and geneticcharacterizations of mutacin II, produced by a group II strain of theoral bacteria Streptococcus mutans was reported. Chen et al. (1999)Appl. Environ. Microbiol. 65:1356-1360; Novak et al. (1994) J.Bacteriol. 176:4316-4320; Novak et al. (1996) Anal. Biochem.236:358-360; Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658.Mutacin II belongs to subgroup AII in the lantibiotic family. Recently,the isolation and genetic characterization of mutacin III from the groupIII S. mutans strain UA787 was reported. Qi et al. (1999) Appl. Environ.Microbiol. 65:3880-3887. The mature mutacin III is twenty-two aminoacids in size, and shows striking similarity with another lantibiotic,epidermin, produced by Staphylococcus epidermidis. Allgaier et al.(1986) Eur. J. Biochem. 160:9-22. The mutacin III biosynthesis genelocus consists of eight genes in the order of mutR, -A, -A′, -B, -C, -D,-P, and T. The genomic organization and primary sequence of mutacin IIIplaces it in subgroup AI with epidermin and gallidermin as its closestneighbors. Applicants disclosed herein the biochemical and geneticcharacterization of mutacin I. Comparison of the biosynthesis genesbetween mutacin I and mutacin III reveal striking similarities as wellas important differences.

[0008] The cloning and sequencing of the novel mutacin I biosyntheticgenes by using information from the conserved sequence derived fromseveral other lantibiotics, and the isolation and purification ofmutacin I is disclosed herein and provides a novel group of antibioticswhich can be utilized as anti-microbial agents against, for example,presently antibiotic resistant microorganisms.

SUMMARY OF THE INVENTION

[0009] According to the present invention, an isolated and purified DNAsequence which encodes a lantibiotic, mutacin I, is disclosed. Thenucleic acid sequence is set forth in SEQ ID No: 1 and the amino acidsequence is set forth in SEQ ID No: 2. Also disclosed are pharmaceuticalcompositions containing mutacin I and methods for their use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended Figures. These Figures form a part of the specification. Itis to be noted, however, that the appended Figures illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

[0011]FIG. 1. (A) The mutacin III biosynthesis genes. The orientation ofthe genes and their relative sizes are shown. mutA is the structuralgene for prepromutacin I, and mutA′ has no known function at present.mutB and -C encode the enzymes for dehydration and thioether bridgeformation of premutacin I. mutD encodes a flavoprotein possiblyresponsible for oxidative decarboxylation of the C-terminal cycteine inpremutacin I. mutP and -T code for the protease and ABC transporter,respectively, which are responsible for the processing andtransportation of premutacin I. (B) Similarity between MutA and MutA′.The middle row shows the identical amino acids and the conserved changes(+). Arrowhead indicates the processing site in MutA. The leader peptideand the mature peptide moieties were determined based on MutA. (C)Effects of mutA and mutA′ mutations on mutacin I production. Cells froman overnight culture plate were stabbed on TH/agar plate and incubatedat 37° C. for twenty-four hours. The plate was heated at 80° C. for onehour to kill the producing bacteria, then an overnight culture of theindicator strain NY101 was overlaid on top of the plate. The plate wasinspected after an overnight incubation at 37° C.

[0012]FIG. 2. Similarity between the mutacin I and mutacin IIIstructural gene. The prepropeptides of mutacin I and mutacin III arecompared using the sequence of preepidermin as a reference. Theidentical amino acids shared by all three lantibiotics are labeled withgray boxes, and the amino acids shared by any of two lantibiotics arelabeled with an open box. The conserved sequence FNLD, which is sharedby all lantibiotics in subgroup AI (29) is underlined. Brackets indicatethe pairs of amino acid residues involved with thioether bridgeformation in epidermin (1).

[0013]FIG. 3. Purification and EIMS analysis of mutacin I. (A) Elutionprofile of the first round purification of crude extract of mutacin I byreverse phase HPLC. One-ml fractions were collected along the course ofelution and tested for antimicrobial activity (insert). (B) Elutionprofile of the second round purification using pooled fraction 6 fromthe first pass as starting material. Fractions 6 and 7 were active. (C)Electrospray ionization mass spectrometry (EIMS) of the purified mutacinI. The mass to charge ratio (m/z) for the doubly-charged molecule (1183)and the triply-charged molecule (788) are labeled. The estimatedmolecular weight was 2364 Da.

[0014]FIG. 4. Biochemical characterization of mutacin I. (A) EIMSanalysis of the ethanethiol-derivatized mutacin I. Peaks 1 and 2 are thedoubly-charged molecule of 1791 Da and 1774 Da, respectively. The1774-Da molecule may be a deaminated form of the 1792 Da molecule. Peak3 may be a deaminated form of peak 4, both of which are singly charged.Peak 5 and peak 6 are triply-charged and doubly-charged molecule of 2719Da, respectively. Peak 7 is a doubly-charged molecule of 2736 Da, whichgives rise to the deaminated form of 2719 Da (peaks 5 and 6). Peak 8 isa singly-charged, deaminated form of peak 9, which has a molecular massof 1793 Da. The expected molecular mass of mutacin I after insertion ofsix molecules of ethanethiol is 2736 Da (2364+62×6), which correlatedvery well with the measured mass of 2736 as shown by peak 7. Addition ofthe two molecular masses of 1791 (peak 1) and 965 (peak 4) results in amolecular mass of 2756 Da, which would correlate well with the intactmodified mutacin I of 2736 Da plus one molecule of H₂O (from breakage ofthe molecule). (B) Proposed structure of mutacin I based on the datapresented in (A) and in FIG. 2. Arrowhead indicates the position wherethe peptide bound is broken in the ethanethiol-modified mutacin I. Thecalculated molecular mass for each fragment is labeled. (C) EIMSanalysis of mutacin III derivatized with ethanethiol under the sameconditions as for mutacin I. The expected molecular mass for fullyderived mutacin III is 2636 (see Table 1), and the measured molecularmass is 2638 from the doubly and triply charged peaks (peaks 2 and 3).The 2620-Da molecule as shown by peaks 1 and 4 are probably thedeaminated form of the 2638-Da molecule. The 2576-Da molecule as shownin peak 5 resulted from addition of five molecules of ethanethiol (seeTable 1).

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides an isolated and purified DNAsequence (SEQ ID No: 1) encoding for a novel lantibiotic, mutacin I,that has been isolated and characterized from Streptococcus mutans CH43.

[0016] Further, the present invention provides the isolated and purifiedDNA sequence for mutacin I designated as mutA (SEQ ID No: 1) andpolymorphisms thereof specific for mutacin I.

[0017] By “isolated” it is meant separated from other nucleic acidsfound in bacteria. By “specific” is meant an isolated sequence whichencodes the protein mutacin I.

[0018] Further, the present invention provides the amino acid sequenceof the mutacin I structural protein SEQ ID No: 2, designated MutA andalso referred to herein as mutacin I, functional variants thereof. Themutacin I protein has a molecular weight of approximately 2364 Da and iscomprised of twenty-four amino acids in its mature form.

[0019] Modification to the nucleic acids of the present invention arealso contemplated as long as the essential structure and function of thepolypeptide encoded by the nucleic acids are maintained. Likewise,fragments used as primers or probes can have substitutions as long asenough complementary bases exist for selective, specific hybridizationwith high stringency.

[0020] Polymorphisms are variants in the gene sequence. They can besequence shifts found between various bacterial strains and isolateswhich, while having a different sequence, produce functionallyequivalent gene products. Polymorphisms also encompass variations whichcan be classified as alleles and/or mutations which can produce geneproducts which may have an altered function. Polymorphisms alsoencompass variations which can be classified as alleles and/or mutationswhich either produce no gene product, an inactive gene product, orincreased levels of gene product.

[0021] The present invention also includes vectors including the mutacinI genes disposed therein. Such vectors are known or can be constructedby those skilled in the art and should contain all expression elementsnecessary to achieve the desired transcription of the sequences. Otherbeneficial characteristics can also be contained within the vectors suchas mechanisms for recovery of the nucleic acids in a different form.Phagemids are a specific example of such beneficial vectors because theycan be used either as plasmids or as bacteriophage vectors. Examples ofother vectors include viruses such as bacteriophages, baculoviruses, andretroviruses, DNA viruses, cosmids, plasmids, liposomes, and otherrecombination vectors. The vectors can also contain elements for use ineither prokaryotic or eukaryotic host systems. One of ordinary skill inthe art will know which host systems are compatible with a particularvector.

[0022] The vectors can be introduced into cells or tissues by any one ofa variety of known methods within the art. Such methods can be foundgenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York (1989, 1992) andAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRCPress, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press,Ann Arbor, Mich. (1995); Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Butterworths, Boston, Mass. (1988); and Gilboa et al.(1986) and include, for example, stable or transient transfection,lipofection, electroporation and infection with recombinant viralvectors. Introduction of nucleic acids by infection offer severaladvantages over other listed methods. Higher efficiencies can beobtained due to their infectious nature. Moreover, viruses are veryspecialized and typically infect and propagate in specific cell types.Thus, their natural specificity can be used to target the vectors tospecific cell types in vivo or within a tissue or mixed culture ofcells. The viral vectors can also be modified with specific receptors orligands to alter target specificity through receptor mediated events.

[0023] The above discussion provides a factual basis for the preparationand use of mutacin I. The methods used with and the utility of thepresent invention can be shown by the following non-restrictive examplesand Figures.

[0024] DNA segments encoding a mutacin gene can be introduced intorecombinant host cells and employed for expressing a mutacin I proteinor peptide. The introduction of the mutacin I expressing DNA can beaccomplished, for example, by the introduction of an organismtransformed with the mutacin encoding DNA to act as a probiotic andproduce the mutacin I in situ to protect against pathogens or otherundesirable organisms. Alternatively, through the application of geneticengineering techniques, subportions or derivatives of selected mutacin Igenes can be employed. Equally, through the application of site-directedmutagenesis techniques, one may re-engineer DNA segments of the presentinvention to alter the coding sequence, e.g., to introduce improvementsto the antibiotic actions of the resultant protein or to test suchmutants in order to examine their structure-function relationships atthe molecular level. Where desired, one may also prepare fusionpeptides, e.g., where the mutacin I coding regions are aligned withinthe same expression unit with other proteins or peptides having desiredfunctions, such as for immunodetection purposes (e.g., enzyme labelcoding regions).

[0025] Pharmaceutical Compositions and Formulations

[0026] Because of the broad spectrum of activity of mutacin I against avariety of microorganisms, mutacin I can be employed to treat multipledrug resistant bacteria such as certain strains of S. aureus which areknown to be multiple drug resistant.

[0027] Pharmaceutical compositions comprising the disclosed mutacins maybe orally administered, for example, with an inert diluent or with anassimilable edible carrier or they may be enclosed in hard or soft shellgelatin capsules or they may be compressed into tablets or may beincorporated directly with the food of the diet.

[0028] A therapeutically effective amount is an amount of mutacin Ipolypeptide, the pharmaceutically acceptable salts, esters, amides, andprodrugs thereof, that when administered to a patient or subject,ameliorates a symptom of the condition or disorder.

[0029] The compounds of the present invention can be administered to apatient either alone or as part of a pharmaceutical composition.

[0030] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0031] For oral prophylaxis, the polypeptide may be incorporated withexcipients and used in the form of non-ingestible mouthwashes,dentifrices or chewing-type gums. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

[0032] The active compounds may be orally administered, for example,with an inert diluent or with an assimilable edible carrier, or they maybe enclosed in hard or soft shell gelatin capsules, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

[0033] The tablets, troches, pills, capsules and the like may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,or gelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compoundssucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

[0034] The active compounds may also be administered parenterally, e.g.,formulated for intravenous, intramuscular, or subcutaneous injection.Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0035] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

[0036] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0037] Dosage forms for topical administration of a compound of thisinvention include ointments, powders, sprays, and inhalants. The activecomponent is admixed under sterile conditions with a physiologicallyacceptable carrier and any preservatives, buffers, or propellants as maybe required. Ophthalmic formulations, eye ointments, powders, andsolutions are also contemplated as being within the scope of thisinvention.

[0038] Intravascular devices, such as catheters, have becomeindispensable tools in the care of seriously ill patients. It isestimated that in the United States alone, 150 million catheters arepurchased each year. However, due to the morbidity and mortalityresulting from catheter-related infections and the high cost of managingsuch complications, the benefit derived from these devices may beoffset. It has been shown that bloodstream infection due to the use ofintravascular catheters (IVC) increased dramatically during the last tenyears. From 1975 to 1977, an estimated 3% infection occurred among IVCusers, while in 1992 to 1993, this rate increased to 19%. The death ratefrom such infection is ˜8000 to 16000 per year, exceeding the death ratefor AIDS. The cost for treating IVC-related infections is ˜132 to 1600million per year.

[0039] Most infections came from the human skin and the hub of thecatheter. Among the infectious bacteria, 40% are coagulase-negativestaphylococci, such as S. epidermidis, and 14% are coagulase-positive S.aureus. The remaining are mostly other gram-positive bacteria such asbacilli and enterococci.

[0040] Prophylactic methods have been developed to prevent IVC-relatedinfections. The first line of treatment is to sterilize the insertionsite with iodine and 70% ethanol. However, compliance with the writtenprotocol is low; only 23% operations follow the protocol. Anotherpreventative measure is the use of catheters impregnated withantibiotics or antiseptic agents such as chlorhexidine and silversulfadiazine. In clinical trials, mixed results were obtained using suchcatheters. In addition to the problem of drug resistance by theinfecting bacteria, the antibiotics coating the catheters can also bewashed away by body fluid, as the attachment of antibiotics to thecatheter surface is mainly through ionic interactions.

[0041] Because of the urgency to solve the problem of IVC-relatedinfection and the growing market for development of catheters resistantto bacterial attachment on the surface thereof, mutacins are anexcellent choice for prevention of IVC-related infections. Mutacin I hasthe following advantages over conventional antimicrobial agents: 1) ithas a wide spectrum of antimicrobial activity against a wide range ofgram-positive bacteria including the multidrug-resistant Staphylococciand Enterococci, the major culprits of IVC-related infections; 2) due toits unique mode of action against the sensitive bacteria, resistance tomutacin has not been observed; 3) mutacin is highly thermostable andworks in a wide range of pH which makes it suitable for use in a widerange of conditions; 4) its hydrophobic nature can be advantageous forcoating the surface of catheters and preventing adhesion of bacteria tothe surface; and 5) because it is produced by a normal member of thehuman oral biota, it is unlikely to elicit immune response from thepatient or has any toxicity to the host.

[0042] Active mutacin I compound can be coated onto intravasculardevices and/or linked to polymers used in the manufacture of thesedevices to be used to prevent infection caused by intravascular devices.The mutacin I compounds of the present invention can be utilized alonein combination with at least one other entity, such as linked to apolymer, for the prevention or reduction of infection associated with avariety of medical devices such as indwelling tubes or catheters,artificial valves, pacemakers, implantable devices, etc., byincorporating, coating, or otherwise combining the active mutacin Icompounds with the materials comprising the patient contact portions ofthe medical devices. The polymer can be a hydrophobic material or matrixthat can be attached to an indwelling device such as a catheter throughhydrophobic bonding or can be tethered to the indwelling device througha molecular linker. The incorporation and/or combination of the activemutacin I compounds may be accomplished by coating the medical deviceswith active mutacin I compounds or by incorporating the active mutacin Icompounds into the structure of the medical device. Because the mutacinI compounds of the present invention are very heat stable, they are ableto withstand the conditions associated with their incorporation into themedical devices. By combining and/or incorporating the active mutacin Icompounds of the present invention into medical devices, both active andpassive infection control can be achieved at sites or for uses, which,in many instances, are highly susceptible or vulnerable to infection.

[0043] The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention. The term “salts” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds of the presentinvention. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Representative saltsinclude the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate,acetate, oxalate, valerate, oleate, palmitate, stearate, laurate,borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate,lactobionate and laurylsulphonate salts, and the like. These may includecations based on the alkali and alkaline earth metals, such as sodium,lithium, potassium, calcium, magnesium, and the like, as well asnon-toxic ammonium, quaternary ammonium and amine cations including, butnot limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. (See, for example, Barge et al., “Pharmaceutical Salts,”J. Pharm. Sci., 1977, 66:1-19 which is incorporated herein byreference.)

[0044] Examples of pharmaceutically acceptable, non-toxic esters of thecompounds of this invention include C₁-C₆ alkyl esters wherein the alkylgroup is a straight or branched chain. Acceptable esters also includeC₅-C₇ cycloalkyl esters as well as arylalkyl esters such as, but notlimited to benzyl. C₁-C₄ alkyl esters are preferred. Esters of thecompounds of the present invention may be prepared according toconventional methods.

[0045] Examples of pharmaceutically acceptable, non-toxic amides of thecompounds of this invention include amides derived from ammonia, primaryC₁-C₆ alkyl amines and secondary C₁-C₆ dialkyl amines wherein the alkylgroups are straight or branched chain. In the case of secondary amines,the amine may also be in the form of a 5- or 6-membered heterocyclecontaining one nitrogen atom. Amides derived from ammonia, C₁-C₃ alkylprimary amines, and C₁-C₂ dialkyl secondary amines are preferred. Amidesof the compounds of the invention may be prepared according toconventional methods.

[0046] The term “prodrug” refers to compounds that are rapidlytransformed in vivo to yield the parent compounds of the above formula,for example, by hydrolysis in blood. A thorough discussion is providedin T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol.14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987, both of which are incorporated herein byreference.

[0047] In addition, the compounds of the present invention can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms for the purposesof the present invention.

[0048] The compounds of the present invention can be administered to apatient at various dosage. For example, the dosage can depend on anumber of factors including the requirements of the patient, theseverity of the condition being treated, and the pharmacologicalactivity of the compound being used. The determination of optimumdosages for a particular patient is well known to those skilled in theart.

EXAMPLES

[0049] Materials and Methods

[0050] Bacterial strains and media. The group I S. mutans strain CH43originated from a Chinese school child as part of a natural historystudy of human caries. Strain CH43 contains a cryptic plasmid similar toother 5.6-kb plasmids within the S. mutans Group I strains. S. sanguisstrain NY101 was used as the indicator for mutacin activity assays. CH43and NY101 were grown on Todd-Hewitt (TH) plate with 1.6% agar (DifcoLaboratories, Detroit, Mich.) unless indicated otherwise.

[0051] Cloning and sequencing of the mutacin I biosynthetic genes.Cloning and sequencing of the mutacin I biosynthesis genes wereperformed exactly as described previously. Qi et al. (1999) Appl.Environ. Microbiol. 65: in press.

[0052] Insertional inactivation. The mutA and mutA′ genes wereinactivated separately by insertion of a kanamycin-resistant genecassette exactly as described for mutacin III. Qi et al. (1999) Appl.Environ. Microbiol. 65:625-658.

[0053] Isolation and purification of mutacin I. For mutacin production,CH43 was grown on TH/agar plate for one day under anaerobic conditions.The cells were then spread on a PHWP membrane with 0.3 μm pore size(Millipore Corp., Bedford, Mass.) on top of a TH plate containing 0.3%agarose. The plate was incubated at 37° C. for two days anaerobically.The membrane was transferred to a new plate for continued incubationevery two days, and the old plate was frozen at −70° C. For mutacinisolation, the plates were thawed quickly in a 60° C. water bath. Theliquid medium was separated from the agarose debris by centrifugationand the supernatant was passed through a membrane with 0.45 μm poresize. Mutacin I was extracted with an equal volume of chloroform. Novaket al. (1994) J. Bacteriol. 176:4316-4320. The precipitate was driedunder a stream of air and washed once with double-distilled H₂O (ddH₂O).The water-insoluble material (crude extract) was dissolved in 6 M ureaand tested for antimicrobial activity by a plate assay after a serialdilution with ddH₂O. One arbitrary unit of activity (AU) was defined asthe highest dilution that showed a clear zone of inhibition of theindicator strain NY101.

[0054] For purification, the crude extract of mutacin I was applied to aSource 15 RPC column and eluted with a fragmented gradient A (0.1% TFA)and B (0.085% TFA in 60% acetonitrile) using a LKB Purifier (AmershamPharmacia Biotech, Piscataway, N.J.). The active fractions were pooledand dried in a lyophilizer. The pellet was redissolved in 0.25% TFA andsubjected to a second round purification using a fragmented gradient ofbuffer A (0.1% TFA) and B (0.085% TFA in 80% methanol). The singleactive peak fraction was collected, dried in a lyophilizer, and used forsequence analysis and electrospray ionization mass spectrometry (EIMS).

[0055] Chemical modification of mutacin I. Fifty micrograms of purifiedmutacin I were dried under vacuum and resuspended in 90 μl of aderivatization mixture consisting of 280 μl ethanol, 200 μl water, 65 μl5M sodium hydroxide, and 60 μl ethanethiol as described). Meyer et al.(1994) Anal. Biochem. 223:185-190. The reaction proceeded at 50° C. forone hour under nitrogen, then stopped by the addition of 2 μl aceticacid. The reaction mixture was dried under vacuum and washed three timeswith 50% ethanol. The pellet was resuspended in 10 μl of 50%acetonitrile with 1% formic acid for EIMS analysis and peptidesequencing by Edman degradation.

[0056] Nucleic Acid accession numbers: The sequence for the mutacin Ioperon has been submitted to Genbank with the accession #AF207710(AF267498), also designated SEQ ID No: 3.

Results

[0057] Cloning and sequencing of the mutacin I biosynthetic genes. Asdescribed previously (Qi et al. (1999) Appl. Environ. Microbiol.65:652-658), while isolating mutacin III biosynthesis genes by PCRamplification using a pair of primers designed based on the conservedsequences among LanA and LanB proteins, the mutacin I biosynthesis geneswere isolated using the same primers. Sequencing of the isolated PCRfragment demonstrated a striking similarity between the mutacin I andmutacin III genes. By chromosomal walking, the major part of the mutacinI biosynthesis operon was cloned and sequenced as shown in FIG. 1A. Itconsists of eight genes in the order of mutR, -A, -A′, -B, -C, -D, -P,and -T, which is possibly followed by the immunity gene mutF (SEQ ID No:12). As in the mutacin III operon, MutR (SEQ ID No: 4) was the positiveregulator for the expression of the mutacin I operon (Qi et al. (1999)Appl. Environ. Microbiol. 65:652-658. MutA (SEQ ID No: 5) and MutA′ (SEQID No: 6) showed strong similarity to each other as shown in FIG. 1B.Insertional inactivation of mutA and mutA′ demonstrated that mutA wasrequired for mutacin I production, while mutA′ was not as shown in FIG.1C. This result suggested that, like mutA in the mutacin III operon, themutA in the mutacin I operon was likely the structural gene encodingprepromutacin I. MutB (SEQ ID No: 7), -C (SEQ ID No: 8) and -D (SEQ IDNo: 10) possibly constituted the modification apparatus forprepromutacin I, and MutT (SEQ ID No: 11) and -P (SEQ ID No: 10) are theABC transporter and protease for transportation and processing ofpremutacin I, respectively. Other gene encoded mutacin I peptidesinclude MutF (SEQ ID No: 12), MutE (SEQ ID No: 13), MutG (SEQ ID No:14), OrfX (SEQ ID No: 15), OrfY (SEQ ID No: 16), and OrfZ (SEQ ID No:17).

[0058] Similarity between mutacin I and mutacin III biosynthesis genes.The overall similarity between mutacin I and mutacin III biosynthesisgenes was ˜94% at the nucleotide level over the 10 kb operon. However,the differences between the two operons were not distributed evenlyamong the different genes. For example, from mutR to the regionimmediately upstream of mutA, the similarity was 99%, while in the mutAand mutA′ coding regions, the similarity was only 89% and 91%,respectively. At the amino acid level, the two MutAs shared 84%identical residues as shown in FIG. 2, and the two MutA's shared 93%identical residues. For MutB and MutC the similarity was 93% and 95%,respectively. An even higher similarity (99%) existed in MutP and -Tbetween the two strains.

[0059] Purification of mutacin I. To biochemically characterize mutacinI, sufficient amount of starting material is required. Applicants' firstattempt to isolate mutacin I from liquid culture failed because nomutacin I was produced in any of the liquid cultures that were tested. Astab culture on TH/agarose plate as described for mutacin III was thentried. Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658. Mutacin Iwas produced on such a plate, however the production level was still toolow for satisfactory isolation. Based on the observation that mutacin Icould be produced on all solid media plates regardless of the mediacomposition, it was reasoned that the production of mutacin I may beregulated by a cell-density mediated control mechanism similar to quorumsensing. (Kleerebezem et al. (1997) Mol. Microbiol. 24:895-904; Suretteet al. (1999) Proc. Natl. Acad. Sci. USA 96:1639-1644). Based on thisrationale, a membrane transfer technique as described in Materials andMethods was employed, which resulted in a high level of mutacin Iproduction.

[0060] Mutacin I was purified by reverse-phase HPLC as shown in FIG. 3.The active fraction (fraction 6) from the first pass (see FIG. 3A) wascollected and subjected to a second round purification using a differentbuffer B and a different gradient (see FIG. 3B). The active fractions(fractions 6 and 7) from the second pass were dried under vacuum andtested for purity by EIMS analysis. As shown in FIG. 3C, mutacin I waspurified to near homogeneity as judged by the lack of significantbackground peaks in the MS chromatogram.

[0061] Characterization of mutacin I by ethanethiol derivatization andMS analyses. The molecular weight of mutacin I was measured byelectrospray ionization mass spectrometry. The mass-to-charge ratio forthe doubly-charged molecule was 1183, and that for the triply-chargedmolecule was 788 as shown in FIG. 3C. Thus the measured molecule masswas 2364 Da. This value was in a good agreement with the calculatedvalue of 2516 Da for the unmodified mutacin I minus six molecules ofwater (108 Da) and one molecule of carboxy residue (45 Da fromdecarboxylation at the C-terminal cycteine residue).

[0062] The primary sequence of mutacin I contained six serine residuesand one threonine residue, all of which were potential sites forpost-translational dehydration. To confirm that there were indeed sixdehydrated residues in the mature mutacin I, an ethanethiol modificationof mutacin I under alkaline conditions was performed. In this reaction,one molecule of ethanethiol could insert into the thioether bridge,resulting in a S-ethylcystein and a cystein, or it could insert into thedouble bound of a dehydrated serine or threonine to form aS-ethylcystein or a β-methyl-S-ethylcycteine. Meyer et al. (1994) Anal.Biochem. 223:185-190; Novak et al. (1996) Anal. Biochem. 236:358-360.Ethanethiol derivatization of lantibiotics has been used prior tosequencing of the other lantibiotic gallidermin and pep5 (Meyer et al.(1994) Anal. Biochem. 223:185-190), and for determination of the numberof dehydrated amino acid residues in mutacin II (Novak et al. (1996)Anal. Biochem. 236:358-360). The expected molecular mass of mutacin Iafter each addition of an ethanethiol molecule is listed in Table 1.TABLE 1 Expected molecular masses of ethanethiol derivatives of mutacinsI and III Expected mass (Da) Mutacin 0* 1 2 3 4 5 6 I 2,364 2,426 2,4872,549 2,611 2,673 2,738 III 2,264 2,318 2,390 2,452 2,514 2,576 2,638

[0063] Quite surprisingly, none of the major peaks generated afterethanethiol modification of mutacin I had the expected molecular mass asshown in FIG. 4A. A very small portion of the molecules showed a pass of2736 Da (Peak 7), which could account for mutacin I plus six moleculesof ethanethiol (2364+62×6); the result of the molecules were all muchsmaller than expected. With close inspection and calculations, theidentity of the small molecules was determined. As shown in FIG. 4B, itappeared that the majority of mutacin I molecules broke into twofragments after the addition of six molecules of ethanethiol. The largerfragment with a mass of 1791 Da was the N-terminal part from F-1 toN-16, and the smaller fragment (965 Da) was the C-terminal part fromP-17 to C-24. This finding was of interest because the closely relatedmutacin III molecule remained intact after the same modificationreaction under the same conditions as shown in FIG. 4C.

[0064] Peptide sequencing of unmodified and ethanethiol modified mutacinI. Comparison of mutacin I and mutacin III revealed that mutacin I hadseven potential dehydration sites (six serines and one threonine), whilemutacin III had six (four serines and two threonines). Interestingly,both mutacins had six ethanethiol additions after ethanethiolmodification (see FIGS. 4A and 4C), suggesting that all serine orthreonine residues in mutacin III were dehydrated. To determine whichserine or threonine residue was not dehydrated in mutacin I, thepurified mutacin I was subjected to peptide sequencing by Edmandegradation. With native mutacin I, Edman degradation was blocked afterthe first F residue, suggesting that the second serine residue isdehydrated. Dehydrated amino acids were shown to block Edman degradationin other lantibiotics. Gross et al. (1971) J. Am. Chem. Soc.93:4634-4635; Mota-Meira et al. (1997) FEBS Lett. 410:275-279; Novak etal. (1994) J. Bacteriol. 176:4316-4320.

[0065] To get a complete sequence of mutacin I, theethanethiol-derivatized mutacin I had to be used.Ethanethiol-derivatization of lantibiotics was shown to allow Edmandegradation to proceed through the dehydrated serine and threonineresidues and thioether bridges in other lantibiotics. Meyer et al.(1994) Anal. Biochem. 223:185-190; Mota-Meira et al. (1997) FEBS Lett.410:275-279. Since the majority of mutacin I molecules was broken intotwo fragments (see FIG. 4) during ethanethiol modification, theC-terminal fragment had to be eliminated to solve the problem of havingtwo N-termini in the reaction mixture. After several trials, theC-terminal fragment was eliminated by washing the reaction mixture with30% acetonitrile. The pellet fraction after 30% acetonitrile washcontained mostly the full-length modified mutacin I and the N-terminalfragment. Sequencing of the pellet fraction revealed the followingsequence:F₁-SEC₂-SEC₃-L₄-SEC₅-L₆-SEC₇-SEC₈-L₉-G₁₀-SEC₁₁-T₁₂-G₁₃-V₁₄-K₁₅-N₁₆-P₁₇-SEC₁₈-F₁₉-N₂₀-SEC₂₁-Y₂₂-SEC₂₃.S-ethylcysteine (SEC) was the product of ethanethiol insertion into thedouble bond of dehydrated serine, or the thioether bridge inlanthionine. The results revealed that all six serine residues in themutacin I molecule were dehydrated, and that T-12 remained as anondehydrated residue. In addition, a closer look at the HPLCchromatogram of the sequencing reaction of mutacin I revealed minorpeaks in the sequence of P-x-F-N-x-Y. This sequence correlated with theC-terminal fragment of mutacin I: P₁₇—S₁₈—F₁₉—N₂₀—S₂₁—Y₂₂—C₂₃—C₂₄. Thisresult corroborated the previous assignment for the two peptidefragments generated during ethanethiol modification as shown in FIG. 4B.

[0066] The mutacin I biosynthesis genes from the group I strain of S.mutans CH43 were cloned and sequenced. DNA and protein sequence analysisrevealed that mutacin I and mutacin III are highly homologous to eachother, likely arising from a common gene ancestor. Mutacin I wasproduced by a membrane transfer technique and purified to homogeneity byreverse phase HPLC. The mature mutacin I is twenty-four amino acids insize with a molecular weight of 2364 Da. Ethanethiol modification ofmutacin I revealed that it contains six dehydrated amino acids.Sequencing of the native and ethanethiol-derivatized mutacin I by Edmandegradation demonstrated that mutacin I is encoded by mutA, and that thesix serine residues in the primary sequence of mutacin I are dehydrated,four of which are possibly involved with thioether bridge formation.Comparison of the primary sequence of mutacin I with that of mutacin IIIand epidermin suggests that mutacin I likely possesses the same bridgingpattern as epidermin.

[0067] A closer inspection of the differences between the homologousgenes of mutacin I and mutacin III revealed that they are not alldistributed evenly. For MutR, -D, -P, and -T, the homology is over 99%between the two mutacins, while for MutA, -A′, -B, and -C, thesimilarity varies from 87 to 95%. The distribution of the variationswithin a protein is not even either. For example, in MutA, the leaderpeptide region was identical between the two mutacins. However, themature peptide region differed by 37.5% (FIG. 2). More interestingly,the sequence of the mature mutacin III is closer to that of epidermin(77% similarity) than to mutacin I (62.5% similarity), while thesequence of the leader peptide of mutacin III and epidermin aredramatically different as seen in FIG. 2. For MutB, -C, -D, -P, and -Tproteins, mutacin I and mutacin III are closer to each other than toepidermin.

[0068] The biosynthesis of lantibiotics involves severalposttranslational modification steps. Chakicherla et al. (1995) J. Biol.Chem. 270:23533-23539; de Vos et al. (1995) Molecular Microbiol.17:427-437; Sahl et al. (1998) Annu. Rev. Microbiol. 52:41-79. The firststep is the translation of the structural gene message into aprepropeptide. The prepropeptide is then modified by dehydration ofserine and threonine residues, and formation of thioether bridgesbetween cysteine and the dehydrated amino acid residues. The prepeptideis then translocated across the cell membrane, where the leader peptideis cleaved off and the mature peptide released to the outside medium.

[0069] One advantage of lantibiotics over classical antibiotics is itsgene-encoded nature, which means that lantibiotics can be altered withease by manipulating the structural genes through mutagenesis. Inreality, however, the number of mutations that can be made is limitedbecause the production of active lantibiotics depends on correctpost-translational modification and processing.

[0070] Mutacin I and mutacin III are closely related to each other atboth the nucleotide and amino acid levels. Comparison of the maturepeptide sequence of mutacin I and mutacin III suggests that they mayalso have the same pattern of thioether bridge formation. Despite allthe similarities, some important differences exist between the twomutacins. For example, ethanethiol modification of mutacin I broke themolecule into two fragments between N-16 and P-17 as shown in FIG. 4B,while the same reaction did not affect the integrity of mutacin III asshown in FIG. 4C. Comparison of the two mutacins revealed that the majordifference is at the linker region (T-12 to P-17), where mutacin I hasthe sequence T-G-V—K—N—P, and mutacin III has the sequence A-R-T-G asshown in FIG. 2A. These different amino acid residues, according to thestatistical figures of Creighton (Creighton, p. 235, in (ed.) Proteins:Structures and molecular principles, W. H. Freeman and Company, NewYork), have different tendencies in forming different secondarystructures in proteins. For example, N-16 and P-17 in mutacin I are morelikely to be involved in forming β-turns, while A-12 in mutacin III ismore likely to participate in α-helix formation (Stryer, p. 37, in (ed.)Biochemistry, W. H. Freeman and Company, Biochemistry, New York). Moreimportantly, N-16 and P-17 are absent in mutacin III.

[0071] In accordance with the possible difference in secondary andtertiary structures, mutacin I and mutacin III have differenthydrophobicity and antimicrobial activity. In reverse-phase HPLCanalysis, mutacin I is eluted at a higher acetonitrile concentrationthan mutacin III, suggesting that it is more hydrophobic than mutacinIII. In antimicrobial spectrum assays with a limited set of pathogens,mutacin III is more potent than mutacin I against Staphylococcus aureusand Staphylococcus epidermidis, while both mutacins have equalactivities against other pathogens such as enterococci, pneumococci, andGroup A streptococci.

[0072] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0073] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentmethods, procedures, treatments, molecules, and specific compoundsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

1 19 1 168 DNA Streptococcus mutans 1 atgtcaaaca cacaattatt agaagtccttggtactgaaa cttttgatgt tcaagaagat 60 ccaacagata ctactattgt ggcaagcaacgacgatccag atactcgttt ctcaagtttg 120 agtttaacag gggtgaaaaa tcctagtttcaatagttact gttgctaa 168 2 24 PRT Streptococcus mutans 2 Phe Ser Ser LeuSer Leu Cys Ser Leu Gly Cys Thr Gly Val Lys Asn 1 5 10 15 Pro Ser PheAsn Ser Tyr Cys Cys 20 3 15567 DNA Streptococcus mutans 3 aaatttgttttttatactaa aagcgggaat gattcaaaac taaaaaagat aaacgaagaa 60 ttgaaaaagtgatataatag cacagaagag ggcctttata atgaaaggag actattttga 120 aagtaaatcaatcaatggaa ttaggtgaac tttatcgaga attaagaatt gctagaggtt 180 tgaagataaaagatatagct tgtaaaaatc tgtccaagtc acaactctct agatttgaaa 240 atggacaaaccatgttggca gctgataaat tgctattagc tatttcggga attcatatga 300 gtttttcggaatttggatat gctttgagcc attatgagga gagtgatttt ttcaaaaggg 360 gtaataagttatcagaatta tatgtccaga aagatatcaa aggattaaaa aagttattag 420 aatttaatgacaatcatgag gtatttgatg tctacaatcg tttaaataaa ttggttattc 480 aagttactattcatttgcta gatactgatt acataatatc agatgatgat aagaattttt 540 taacaacttatctatataat attgaagagt ggactgagta tgaactttat atctttggaa 600 atactatgtctatattgtca tctgatgatt taattttttt gggaaaagct tttgtagaac 660 gtgataagttgtatatatct cttcctagtc ataagaaaaa tgcagagtta acttttttaa 720 atttaatcttaattttgctt gaaagaaaaa aattatatca agcaatctat tttgtagaga 780 atttagagaaattattaaat taccaagata tgtttgcaat aacattttta aaatttttaa 840 aaaaaattattacttacttt catgataagt cagtagatat gtctgaatta gaacattata 900 ttaatatagttgaagaaata aatcctacga ttgcttcaat tcttaaatct aatttgaatc 960 agcttttatcaagttttagc cattaaagcc atcttgataa attttatatc tttcatattc 1020 attaaatgtggagataatga aaaagcaacg gttatgctat cgctgctttt tttgtgatta 1080 gaagctatgttatcatggag ttatagtaat gaaacatagt gacagttcat catttcttat 1140 tataaaagtggtaataagag aagtggtaaa caaagagtta gtaaaataat acgtttaacc 1200 ataatatttcctcctttaat ttattataag attcaaaaag gtaatattcc tatatttgca 1260 aatatgggataaaataattt taaaaaagca gatttgcaat tttaaaaaaa atagaggcta 1320 atggtggtattatattattg taaatatatg tttactcagt aatagtgatt tactattaca 1380 acagattttgttgttatctt agatatttct gctagcatta gttatctgta gatgtactac 1440 ttaataagtatataattata attatataat aactattatc agattaccgt taaaagtttt 1500 ctgatatgcttctactgaac aatttacgtt cagttacaca catgaaaaag gaggatatta 1560 tgtcaaacacacaattatta gaagtccttg gtactgaaac ttttgatgtt caagaagatc 1620 tctttgcttttgatacaaca gatactacta ttgtggcaag caacgacgat ccagatactc 1680 gtttctcaagtttgagttta tgttcattag gatgtacagg ggtgaaaaat cctagtttca 1740 atagttactgttgctaagtt gtacaaaaga tttagattgt gtcgcatgtc agcggcacaa 1800 tcttttgatattagagatat taaatatgtt aaacacacaa ttattagaag tccttggtac 1860 taaaacttttgatgttcaag aagatttatt tgagtttaat ataacagata ctattgtact 1920 gcaggttagtgatagtccag gtactcatag taaagtgggt agtttcagta tctgtcctcc 1980 tcgaaagacctccgtcagtt tcaatagtta ctgttgttaa ctataaatta tacttaaatt 2040 gataggaaacttggtcatga cattatcata tgttgatatt ggaagagaat caaatttata 2100 aagacaattaaatctaaatt tgatgaatat ttagatgaat tattactagg ttgacagtca 2160 tgttaggagaagagatgaac gattttcaat ttcaagatta ttttatgtac agaaaaccat 2220 taggcaacttttctaatttt cttagtataa ctgatatgat ggatcctatt gaattattac 2280 ataatgatccgatatttgct gaaggggtat atttggcttc cccatctctt agatcatcta 2340 taaataaattagagaatcag attgcaagta ctaaggaaaa aaagaatgca aaagagacta 2400 tttttcaatactatgcccgt tataacacga gatcaactcc gtttggcttg ttttcgtcca 2460 tcggaataggtggtttttcg aaccacccta ggaaagagaa atcttgttat gaaaaatctg 2520 ttaatgttgatcttttttgg gcttataaag tagcagataa actagaaagt atgcctgaaa 2580 ttttaaatactttaaaagta gttgctaata atgctttgca aaagtcaaat gatttttggc 2640 ttttagatacacgaagtcat tttggactta tgaattcacg ttcagatatt cgtgaggaca 2700 ttacagttaagtctaatcag cttatagatt atgttattaa ttgcacagaa gaaccaatta 2760 gctatcaaacattaattgat gatattgccg agaaattctc tcaatctagt gatgatgtaa 2820 aagaatatttgcaaacatta attaaagagg agtttttaat aactgaattg aaatttagtt 2880 tgattgatgataatcctttg gattggttta ttaatatttt agaaagagat caaaataact 2940 cagaattacttgaaaagttg actgaaataa aggcaatgat tcaagattat actgaccgta 3000 acataggtgaaggtaacaat tcgattttag ctctagaaaa taagatgagc caaatagtaa 3060 aagccaacgcatacctgcga gttgatcttt atgatcatgc agagctgaag ttagcgcaac 3120 ataccaagagttctcttcag aatattttga aagtactaag ttctttttcg tcagctgtta 3180 atagtcaaaaagaaattaaa aattatcatg agaaatttat tgccaggtat ggatacgagc 3240 agttagtacctcttcaatta cttttgaatt ctactagtgg acttggtttt ccaaaagggt 3300 atagtcaaacagaagtttct aaacaaaata atgaagatag taaaaatcaa aaaataatag 3360 aatttttacagagaaaattt gaaaaagctt taagagatgg taaagaaatt attttgagtg 3420 atgatgatttaaaagattta aattttgaca cggaacagca aatatcagga gaattatatt 3480 gtttctacaattttaaaagt aaaaagctag aggttagtag tttaggtgtc tcacagatgc 3540 ttggaaatacttttggacgt ttccattcta aattgccgaa tacgatagtc acaaaaaatg 3600 taaataagacgaaagaaatt tttactgagg cttatccaaa tactattatt actcaattaa 3660 atgaagtgccatattttggg agaggtggca atattatgat tagtaatagc cttaaaagtc 3720 accagttggaattgaggaac tatactacta aaaaagagat gagtatcaat gatatttatg 3780 tacgtgcaaccagtgaggag ttatattttt attctaagaa atatgagaaa agagttattt 3840 ttgtgatgaataatatgttt aattatataa atggttctaa actcttacgt tttttactag 3900 aagtttcaaattctgatttt caaaatatta ccccgattac gcttggtagt ctggattctt 3960 ataatcatgtgcccgctatc atttataaag atattattat taaaccggaa acatggaaca 4020 ttagaaaatctgaagctaag actttagatt ctctcaaaaa ttggctaact aataataatg 4080 ttccgccttttgtacggatg aaatatactg atcaaattat ttatttagat ttgagtcgga 4140 ctattgatttaactatgcta tttcagagta tcaaaaaaca tagcttcata caattattag 4200 atgttcattcagtatgtaca aacgatacgg agattttaga attagttgtt ccttttacaa 4260 gaagtgatgttaacgctcac cagatttatc attatgctca gaatatttat actttggagg 4320 attcaggtagtaaagaaaaa tatttttacg ctaaaattta tgtgaataaa caacgacaga 4380 cctctttcctacaaaaagag tatcctttat tattaaaata tttgaaactc ccagaaaact 4440 tacaatggttctatattaga tataaagatg atggaaaaga cagcatacgt ctcagaatca 4500 gatatgtagaagataaacaa ttagttcaac tttattcacg ctttatagag tgggcaacaa 4560 aagcacggaaaaatatccaa atttcaggtt atgaaattag tgaatatatc cctgaatcag 4620 caagatatggagggaaaaaa tattcttcaa ttattcattc ttttttctat tatgatagta 4680 ttttggatttgcttttacag aagaaagcag aacaaactat tgaagtaaga acatctctca 4740 gtattattcgtatgttttta atgatgaaat taagcttaca agaccagcag aaactcataa 4800 agaatttatttgatggaaaa cataaactta aatatgaaaa agaatatcat aattcaataa 4860 gtttattacttgacaattta tgtacaaaaa atcagacaga tgaagctgat attttctgtg 4920 taatgaatatgaaaaaaatc actgaaaaaa ttagctcagt tcttaaacaa aaggacttaa 4980 caacagattggcagagaatt ctaggaagtt taattcatat gcgatgtaat cgagtatatg 5040 gaattaacagtgagttagaa agaaaaacaa tgtttattgt tgacaaagtt attaattcaa 5100 aaagatatacggatatgttt ttggaggtgg gtaatgagac aaagtaaacg tgtcgaaaaa 5160 attaaagatattctaactga gcaaacttat ttattcgatt atcaagaaat attaaaaaaa 5220 gtcagtcaagcaaaacaaac agatttttgg aatttacttt ccttatcttc gggaataact 5280 tctttattaatattttatca agagtatgag aatttagaag gagtaaactt aaagcagcaa 5340 aagcagtcattaattgggct tataagtcat tatattaatc aaatagcaga gaaatcctct 5400 ttatttgatggtttagctgg ggtaggtttt gctattaatt atatctctaa taacggtaaa 5460 tattatcaaaaacttcttga acagattgac aacagactcc gtcagaatat tgaacggaac 5520 cttgtcaactataagaatga ggaatatgca aatcctatga attatgatgt agtttctgga 5580 aatgctggagtagctcgcta cttgatggaa agagaatcct ctgaagattg gcgaatagtt 5640 gaaatgattttagaaacatt ttataaagct ttagagcaag gctggcgagt acagtcaaaa 5700 tatcaatttctagagtctga aaagcagtat tatctagaag gaaatataaa tttcgggttg 5760 gctcatggaatattaggacc tgcgacaatt atggctcttt atcaacgaag agaaccacaa 5820 aatacaagaaatgctgagaa gcttcaagaa acttatcgac taataaaaag atacgcccag 5880 gtaagagatgaagggttacg atggccaata cgatatgatt tgtttcgtaa agagggttct 5940 tttatattacgaaatggttg gtgttatggc gagaatggca tttataatac actttttctt 6000 atgggaaaagtactctcaaa tcaggagatt tgtgaaactg ctcagaaagt tataccatcc 6060 atcataaaagatgattatga gaaaatggaa agtccaacat tttgtcacgg gtttgctgga 6120 aaagcaaatttctttcttct gcaatatcaa agaactaaag aatcaatatt tttagttaaa 6180 gcagaagaagaaattgataa aatattaatt gtgtacaatt ctgaaaatat gtttggattt 6240 aaagatatagaagataatat tgataatact ggagagagat taacttattg ggataatttt 6300 ggtcttcttagtggaactgt tggtgttcta ttagttttga tggaatattg taatattgta 6360 aatgccggaaaaattgcaga gtggaataaa atttttcttt tgacttaatt aactgaacgg 6420 agaaataattatggaagaac aaaatataga gaaaaaaatt ctcttgtgcc taacaggttc 6480 tggagcattgttagggatag ctgaatatat tacgtttttg actgtgcgct ttaagcatgt 6540 tcgagttattgtctctgata atgctgcgaa gatgcttcct gttgctgcta ttacacaatt 6600 gtgtgagaaagtgtatactg atgaagtttc ctttacagat aagcaaaaga atcacatagc 6660 tttaactcgctgggcagaca taacagttgt cttacctgct acagcaaata taattggaaa 6720 agttgctaatggtattgcag ataactttat gacaacaact cttctttctt ctagcaagcc 6780 agttttaatttatccttgca tgaataatat tatgtgggaa aatccagtag ttcaaaaaaa 6840 tgttgaagttttatctggaa cccaatataa ggtaattgtt ggacaagaat cagaatcttt 6900 tgaattagccagtggaaaga tgaaaaagaa tattgcaatt ccaagtttgg atgaattgca 6960 acgagttgttttagaaaatt tacaagaaga gaggtaagag tatgaagaag aaaggattac 7020 tagtaataatctttctaact ttctttttct tttatcctaa agctaaagct gctgaatata 7080 caattatatcaaataatagt gaacaaactg ttaatgactt gaataattta ggagttacag 7140 tcaatagccatattgcggaa attggatata ttgaagctca aggagatgtt aacattgatc 7200 agattaaaaagctgtcaaat attcaaagta tccagaatat ggctgataca tcacagaata 7260 tcacgactagagttccttca acatatatta accagacaat acaattgcct cagctttttt 7320 cttatcagtgggatatgcaa aaaattacta ataatggtgt ttcatattca ttaaataaag 7380 aaaatcgaaaaaatgtaaca gttgctttag ttgattctgg gattgatgta gaccataatg 7440 cttttacaggaatgattgat agtcgttcaa aaaattttgt gcctgctgga ggatatgata 7500 atagtgaaagcagtgaaact ggaaatatta atgatattga tgataaaaaa ggccatggaa 7560 cagcagttgctgggcaaatt gctgcaaatg gtcaaatctt tggtgtgtcc ccaggaacga 7620 accttcttatctatagagtt tttggaaaat caaaatcaaa ggagtgctgg attttaaaag 7680 caattattgatgcaacaaat aacggtgcta atgttattaa tctaagtttg gggcaatata 7740 ttaagattcctaatggtgat atttgggagt ctgccgaagc attaggatat aagtttgcca 7800 ttgattatgccacaagacat aatgtcattg ttgtagcagc cacaggtaat gatggattaa 7860 gtgatgacaacggagaggtt aaaacttatt ataatagtca gcattcagga caagatatgt 7920 ctcaaaatgacacggttgaa gattatcctt ctgttttacc taatgctatt gcagttggct 7980 cttctgataataataatcaa agatcatctt ttagtaatta ctataatcaa tatcaggaca 8040 attttattttggctcctggt ggtggaacaa ctttactaga ccaatatggt caagaagagt 8100 ggtataatcagaaacttttt atgaaagaac aagtcttatc aacaagtaat aatggaaatt 8160 atgattatgcagatggtact tctatttcaa caggaaaagt ttctggagag cttgcagaaa 8220 ttattagtaactaccatctt caaggagatt cttcaaaagc tagaagtatt ctactaaatc 8280 aagttaattatactagtgat ggttataaag aaataagcac ttacaaagct ttgcgaggtt 8340 actaaatgaagtggttagaa gttttgcaaa ttagtaaaaa agaaaaaatt ctttatctta 8400 ttggttgtatattttcaatt atgacaggct taattactct acgaatcacc tacttactta 8460 agaatttagttgacagcaaa tcgtctttta ataatttgtt cttgtttctt gttttgggat 8520 tagttctttttatcatagat gctggttcac agtatctaat ttcattgatt ggtaatcaag 8580 tagtgtttaacagtcgaaat aatatttgga aaaaaatttc tgattggaca gatagtaaag 8640 atgattcttctgaaatggca ggccacctta ttaatgatag tgaactgata gaaaatttta 8700 taatttctactattcctcaa tcaataaatt cagttattgt tggatcagga tccttagtta 8760 tgctatttgttattaatagt aaaatgtctt tagaagttat agggatttgc ttgcttttat 8820 tgttcattatgcaacccttt tctagaatat taagcaaaat aagtaaaaga atccaggaag 8880 acaaagctgaacttattaat attgcctcac agttgagagg acaagtcaaa acaataaaaa 8940 gctataatgctcaagattat gcctttcaaa aatttgatga gcaaaatcgc caattatttc 9000 aagatatcttaaatagaata aaaattttta gcatttactc tcctttttta aatatcttaa 9060 ttctttttatgattataatt gttgtttggc taggaaatac agaagtacgt tcaggaaatc 9120 tcactgtaggttcagcaact atttttgttg tttatatgac acaattaatt aatccaatta 9180 tgcaattatcacaattagtt gctcatatgg ggatgcttaa tggcggcgtg gaacgtcttt 9240 tggagtataatcaagctatt ccagaaaaaa atggaatcaa gaaaattgat gaaataatta 9300 atatcgcgtttgataatgtt tcatttgctt atgataacca agaaaatatt attgaaaatg 9360 tgaatttaacttttcaaaaa ggtacttata tttccattgt tggtgaaagt ggagttggga 9420 aatcaaccttacttgatctt ttagaacata attatgtacc atcaaaagga cgaatcttaa 9480 taaacggaatagacttagaa gaattgaata ttaagacttt gcgaaataag ataagctatg 9540 tatctcaagaaccaacaatt ctttctggga caattcgtga actattagac tttaatcagc 9600 aacagcatacagaaactagt ctttggaatg ttcttgatac tgtagaatta tcagaactta 9660 ttagaaatttacccgcgaaa ttagattcta aggttgatga atatggtggt aacctctctg 9720 gaggtcagatgcaacggatc tcacttgcaa gaggattact gaaagcagga gatgttttat 9780 tattagatgaatcttttgcc aatattgatg aagagacttg tcttaaaata aaattaaaaa 9840 ttgctgcttatgctgaatca cacaagcaaa ttgttattga agttattcat aatctaaata 9900 gaataactcccagtagtatc gtttaccgat tggctgataa aaaactagaa attttgagga 9960 gcggattttaatagaaaagt cgaagaaatc tgagtaaaag atcagtttct ggtcgaaaat 10020 taaatattgtgatatataaa taagcttaaa atcaatattc ctaataattt gattttaagc 10080 tttttactatttgatgagtt tttactcaag atcttttgat tttcctgata aagtccttaa 10140 atttgttttttatactaaaa gcagaaaagg aggatatcat aatggattat atgctagaga 10200 cgaaaaatttaactaaacag tttggtaagc aaacagcggt taaccaattg aatttgaaag 10260 ttgaacgtcattcaatttat ggtttgctgg ggcctaatgg ttccggcaaa tcaacaacac 10320 ttaaaatgattactggaatg ctaagaaaga catctggtca cattcttata gacggacacg 10380 attggagccgcaaggattta gaaaatatcg gggctctgat tgaatcaccg ccgctttatg 10440 aaaacctgactgcgcgtgaa aatttaaagg taagaacctt gatgctgggt ttacctgata 10500 gtcgcattgatgaggtttta aaaatagtgg atctaaccaa cacgggtaaa aaaagagcag 10560 ggcaattttctatgggcatg aagcagcgtc tgggtattgc tatcgcactt ttgaactcac 10620 ctcaacttttgattctggat gaaccgacta atggacttga tcctattggt attcaggagt 10680 tgcgtaatcttattcgttcc ttccctacac aaggaattac agttattatt tccagtcata 10740 tcttatctgagattcagatg acagcggatc atattggtat cattgctaat ggcgtactgg 10800 gttatcaggatagaattcac caagatgaag acttggaaaa actttttact gatgtggtta 10860 tgagataccgaggaggtgag tgatatgctg ggcatgtttc aggcagaaag gttaaaactg 10920 aagcgaagtatggcgaagaa gttactagtt tttgccccca taatagctat tttatatggt 10980 tttatagcacctgtggggta tttagtaaat aatgcttata attggtggta tgtcatgatt 11040 tttccagggctgctaacctt atttgctgct ttaataaata cttacgaaga aaaaaagctg 11100 cattatcgagcagtgtttcc tttgcccatt tctttaagaa aattttggtt tgaaaaaatt 11160 tttataactgtttattatct taattttagt aatggagtac tttggataat tacagtatta 11220 ctgaatacttttattttacc aaattatgga aaagactata cttatactgt tggagaatta 11280 gcactagcttctttggttat aatagttact acactttggc aaattccatt ttgtctgtgg 11340 ctgacaaaaagaatcggttt taccataacg ttgataatta atttaatgag taatttcatt 11400 ttgggagttgtttttgcaac tacttcctgc tggtggcttt gtccatatag ttggggaata 11460 cgattaatggtacccatttt aaaaatacta ccgagtggtc taaaggcagg tatagcagga 11520 gctccatcattgccaacaag tttttggagt atcgttatta gtttgtgttt agcggttatc 11580 ttatttgttagtttgacagt tttgagtgca tcttggtttg aaaaacagga agtgaaatga 11640 tgattgatttattaaaagca gaaaatgtaa aataccgtca tactttttta ccatggttac 11700 acctgattttacctgttact acagctattg ttgttattgt ttatgggcta atgacgccga 11760 ctcactcttgggctgatatt actggtggtt acttagaact attgggtata agttttccaa 11820 ttgtcattgctgttatttgt gggaaatcag ttggactaga agtagaggct ggtcaatttc 11880 aagttatgttagcaattaag caaaggaact tgatattttg tatcaagtta ttgaatttgc 11940 tcattttagaacttttttca actctattag ctataggaat ttatggatta atttatcaat 12000 taagtaataaacatttgata ttttatggat atgctgtaat tttactaaca gcttcaatgc 12060 tcattctttatctgattcac ttagttgtag tatttttgtt tggcaatagt gctaatattg 12120 ggttggggattgctgaatct ttactatctg ctttgctctt gacaggttta ggagatggta 12180 tctggcaatttattccttgt gcttggggta ctcgcctaat gggtacctta ataaatctgt 12240 ggtattactctgggcacagc ttatttttta agcaacagct tttaatttgg ctggaagtcg 12300 cagttccactaactttaatg gctttaatcc ttagtataat ttggttcgac agatggcaag 12360 gacgtagcagtgatgaataa aggaaaaagg agaactttca aacatgacct atattggtgt 12420 tagtcatctcaaaaaggtgt ataaaactca ggaaggcctc actaacgaag cgttaaaaga 12480 tattacgttctcagttcaag aaggggaatt tattgctatt atgggtgaat ctggctcagg 12540 gaagtcaactctccttaata tcctagcttg tatggattat ccaagtagtg gtcatatcat 12600 cttcaataactatcaattag agaaagttaa agatgaagag gctgctgttt ttagaagtcg 12660 gcatattggttttatttttc aaaatttcaa tcttttaaat atcttcaata ataaagacaa 12720 tctgttgataccagttatta tttcgggaag taaggtgaat tcctatgaaa aacgattacg 12780 tgatttagctgctgttgttg gtatagaatc tttgctatct aaatatcctt atgaattatc 12840 tggaggtcaacaacaaaggt tagctattgc cagagcttta attatgaatc cagacttgat 12900 attggccgatgagccaacag gacaattgga ctctaagact tctcagcgaa tcttgaattt 12960 gttgtctaacatcaacgcta aacgaaagac aattctaatg gtgactcata gtcctaaagc 13020 tgctagttatgcaaaccgag ttctttttat caaggatggt gttattttca atcaacttgt 13080 tcgtgggtgtaaatccaggg aaggcttttt agatcaaatt attatggctc aggccagtct 13140 gtaggaggttgtcctgatta tgtttttacc caaaatttcc tttcataatc ttattgtaaa 13200 taaatcattaaccttacctt attttgctat tatgaccatt tttagtggtt ttaactatgt 13260 tttgattaattttttaacca accctagttt ttataacatt ccaacagcta ggatactgat 13320 tgatattcttatttttggtt ttatcttaat ttcattactg atgttgcttt atggtcgcta 13380 tgccaatcgttttataagtg atgagcgtaa tagtaatatg ggaatttttc tcatgttggg 13440 aatggggaaaaagcaattat taaaaataat ctatttggaa aagttatatc tttttacagg 13500 aacgttttttggaggtttaa tctttggttt cgtatacagt aagatatttt ttctttttat 13560 cagaaatctaattgttattg gagatgtcag agaacaatat agcttaacgg ctattagttg 13620 gctacttattcttacttttt ttatttattt tattatttat ctatcagagt accgattatt 13680 aaaacgtcaaagtatcacgg ttatttttaa tagcaaagct aagcgtgata atcctagaaa 13740 aactagtgtttttgttggac tttttggact ttttgccctg ttaatgggat atcattttgc 13800 tttaacaagtcccaatgtca caaccagttt cagccgtttc atttatgctg cctgcttagt 13860 tactctaggtattttttgca cgttttcgtc aggtgtgatt atgttactga ctgtcataaa 13920 gaagagaagagctatctact ataatcaacg gcgctttgtt gtgattgcta gtttatttca 13980 ccgtatccgcagtaatgctc tgtctttggc gactatctgt atttttagca ccgctacctt 14040 agttagtttatctgtcttag ctagtctcta tcttgcaaag gacaatatgg ttcgtctttc 14100 aagtcctagagatgttacgg tgctatctac aactgatatt gaaccgaatt taatggacat 14160 cgctacaaaaaatcatgtta ctctaactaa tcgccagaat ttaaaggttt ctcaatctgt 14220 ttatggtaatatcaaaggaa gtcatttgtc agttgatcct aatggcggta tggctaatga 14280 ttatcaaataacagttattt cattggattc ttttaatgct tctaataata cccattatcg 14340 tttaaaaaatcatgaaattc tcacctatgt ttcaaatgga gcagctgctc cctctagcta 14400 tacaactaatggtgttaaac taaccaatgt taaacaaatt aaaaggataa actttatttt 14460 ttctccgctacgctctatgc agcctaattt ctttataatt actgacaatc gagaaataat 14520 tcagactattttgaaagagg agctaacatg gggaacgatg gcaggctacc atgttaaagg 14580 aaaaaaaatgaatcagaaag atttttatga tgagcttgag actactaatt tcaggcaatt 14640 tagtgctaatgtagtttcaa taagacaggt caaatcaatg tttaatgctt tatttggcgg 14700 tttactctttgttggtatta tttttggaac tatttttgca attttgacag ctataactat 14760 ttattatcaacagctttctg aaggaattcg agaccgagat gattataagg ccatgataaa 14820 attaggtatgacaaataaaa ctattcaaga cagtattaag gttcaaataa actttgtttt 14880 catcttgcccattgcttttg ccctattaaa tctcatcttt gcacttccta ttttatataa 14940 aataatgacaacttttggat ttaatgatgc aggactattt ctaagagctg ttggaacttg 15000 tctgattgtttaccttttct tttattggtt tatttgtcat tgcacatcca aactatatta 15060 tcgtttaatatctaaaaaat agaggagttt atattatgcg tattgtaagt tcattggtat 15120 cgcttttattgactatcttt tggatttttg ctatagcttt tatcccaatt ggagaccaga 15180 atagttttaataaaccagaa atgtggttct ttgttttttt cgctattatt atttatagta 15240 ttgttataataagcgattat tatctaaaga gctttaatct tttgaaagtt tatcaaattt 15300 tagttttgtttattagcata ctgtgtgctc tttgtggttt atcactaact gctttaggat 15360 tgaaagtattcactttagct attggaattg ttagtcttgt taatacaatt atttatttct 15420 ttttcgctaataaaaaagat aatgttgaat aaaatatgtt atcctagtga aggaggtttc 15480 ctagaatgacccgtattttg gtaattgatg atgatgcaga tattttggct ctgataaaaa 15540 ataccttgcaactgcaaaac tatctgg 15567 4 289 PRT Streptococcus mutans 4 Leu Lys ValAsn Gln Ser Met Glu Leu Gly Glu Leu Tyr Arg Glu Leu 1 5 10 15 Arg IleAla Arg Gly Leu Lys Ile Lys Asp Ile Ala Cys Lys Asn Leu 20 25 30 Ser LysSer Gln Leu Ser Arg Phe Glu Asn Gly Gln Thr Met Leu Ala 35 40 45 Ala AspLys Leu Leu Leu Ala Ile Ser Gly Ile His Met Ser Phe Ser 50 55 60 Glu PheGly Tyr Ala Leu Ser His Tyr Glu Glu Ser Asp Phe Phe Lys 65 70 75 80 ArgGly Asn Lys Leu Ser Glu Leu Tyr Val Gln Lys Asp Ile Lys Gly 85 90 95 LeuLys Lys Leu Leu Glu Phe Asn Asp Asn His Glu Val Phe Asp Val 100 105 110Tyr Asn Arg Leu Asn Lys Leu Val Ile Gln Val Thr Ile His Leu Leu 115 120125 Asp Thr Asp Tyr Ile Ile Ser Asp Asp Asp Lys Asn Phe Leu Thr Thr 130135 140 Tyr Leu Tyr Asn Ile Glu Glu Trp Thr Glu Tyr Glu Leu Tyr Ile Phe145 150 155 160 Gly Asn Thr Met Ser Ile Leu Ser Ser Asp Asp Leu Ile PheLeu Gly 165 170 175 Lys Ala Phe Val Glu Arg Asp Lys Leu Tyr Ile Ser LeuPro Ser His 180 185 190 Lys Lys Asn Ala Glu Leu Thr Phe Leu Asn Leu IleLeu Ile Leu Leu 195 200 205 Glu Arg Lys Lys Leu Tyr Gln Ala Ile Tyr PheVal Glu Asn Leu Glu 210 215 220 Lys Leu Leu Asn Tyr Gln Asp Met Phe AlaIle Thr Phe Leu Lys Phe 225 230 235 240 Leu Lys Lys Ile Ile Thr Tyr PheHis Asp Lys Ser Val Asp Met Ser 245 250 255 Glu Leu Glu His Tyr Ile AsnIle Val Glu Glu Ile Asn Pro Thr Ile 260 265 270 Ala Ser Ile Leu Lys SerAsn Leu Asn Gln Leu Leu Ser Ser Phe Ser 275 280 285 His 5 65 PRTStreptococcus mutans 5 Met Ser Asn Thr Gln Leu Leu Glu Val Leu Gly ThrGlu Thr Phe Asp 1 5 10 15 Val Gln Glu Asp Leu Phe Ala Phe Asp Thr ThrAsp Thr Thr Ile Val 20 25 30 Ala Ser Asn Asp Asp Pro Asp Thr Arg Phe SerSer Leu Ser Leu Cys 35 40 45 Ser Leu Gly Cys Thr Gly Val Lys Asn Pro SerPhe Asn Ser Tyr Cys 50 55 60 Cys 65 6 64 PRT Streptococcus mutans 6 MetLeu Asn Thr Gln Leu Leu Glu Val Leu Gly Thr Lys Thr Phe Asp 1 5 10 15Val Gln Glu Asp Leu Phe Glu Phe Asn Ile Thr Asp Thr Ile Val Leu 20 25 30Gln Val Ser Asp Ser Pro Gly Thr His Ser Lys Val Gly Ser Phe Ser 35 40 45Ile Cys Pro Pro Arg Lys Thr Ser Val Ser Phe Asn Ser Tyr Cys Cys 50 55 607 990 PRT Streptococcus mutans 7 Met Asn Asp Phe Gln Phe Gln Asp Tyr PheMet Tyr Arg Lys Pro Leu 1 5 10 15 Gly Asn Phe Ser Asn Phe Leu Ser IleThr Asp Met Met Asp Pro Ile 20 25 30 Glu Leu Leu His Asn Asp Pro Ile PheAla Glu Gly Val Tyr Leu Ala 35 40 45 Ser Pro Ser Leu Arg Ser Ser Ile AsnLys Leu Glu Asn Gln Ile Ala 50 55 60 Ser Thr Lys Glu Lys Lys Asn Ala LysGlu Thr Ile Phe Gln Tyr Tyr 65 70 75 80 Ala Arg Tyr Asn Thr Arg Ser ThrPro Phe Gly Leu Phe Ser Ser Ile 85 90 95 Gly Ile Gly Gly Phe Ser Asn HisPro Arg Lys Glu Lys Ser Cys Tyr 100 105 110 Glu Lys Ser Val Asn Val AspLeu Phe Trp Ala Tyr Lys Val Ala Asp 115 120 125 Lys Leu Glu Ser Met ProGlu Ile Leu Asn Thr Leu Lys Val Val Ala 130 135 140 Asn Asn Ala Leu GlnLys Ser Asn Asp Phe Trp Leu Leu Asp Thr Arg 145 150 155 160 Ser His PheGly Leu Met Asn Ser Arg Ser Asp Ile Arg Glu Asp Ile 165 170 175 Thr ValLys Ser Asn Gln Leu Ile Asp Tyr Val Ile Asn Cys Thr Glu 180 185 190 GluPro Ile Ser Tyr Gln Thr Leu Ile Asp Asp Ile Ala Glu Lys Phe 195 200 205Ser Gln Ser Ser Asp Asp Val Lys Glu Tyr Leu Gln Thr Leu Ile Lys 210 215220 Glu Glu Phe Leu Ile Thr Glu Leu Lys Phe Ser Leu Ile Asp Asp Asn 225230 235 240 Pro Leu Asp Trp Phe Ile Asn Ile Leu Glu Arg Asp Gln Asn AsnSer 245 250 255 Glu Leu Leu Glu Lys Leu Thr Glu Ile Lys Ala Met Ile GlnAsp Tyr 260 265 270 Thr Asp Arg Asn Ile Gly Glu Gly Asn Asn Ser Ile LeuAla Leu Glu 275 280 285 Asn Lys Met Ser Gln Ile Val Lys Ala Asn Ala TyrLeu Arg Val Asp 290 295 300 Leu Tyr Asp His Ala Glu Leu Lys Leu Ala GlnHis Thr Lys Ser Ser 305 310 315 320 Leu Gln Asn Ile Leu Lys Val Leu SerSer Phe Ser Ser Ala Val Asn 325 330 335 Ser Gln Lys Glu Ile Lys Asn TyrHis Glu Lys Phe Ile Ala Arg Tyr 340 345 350 Gly Tyr Glu Gln Leu Val ProLeu Gln Leu Leu Leu Asn Ser Thr Ser 355 360 365 Gly Leu Gly Phe Pro LysGly Tyr Ser Gln Thr Glu Val Ser Lys Gln 370 375 380 Asn Asn Glu Asp SerLys Asn Gln Lys Ile Ile Glu Phe Leu Gln Arg 385 390 395 400 Lys Phe GluLys Ala Leu Arg Asp Gly Lys Glu Ile Ile Leu Ser Asp 405 410 415 Asp AspLeu Lys Asp Leu Asn Phe Asp Thr Glu Gln Gln Ile Ser Gly 420 425 430 GluLeu Tyr Cys Phe Tyr Asn Phe Lys Ser Lys Lys Leu Glu Val Ser 435 440 445Ser Leu Gly Val Ser Gln Met Leu Gly Asn Thr Phe Gly Arg Phe His 450 455460 Ser Lys Leu Pro Asn Thr Ile Val Thr Lys Asn Val Asn Lys Thr Lys 465470 475 480 Glu Ile Phe Thr Glu Ala Tyr Pro Asn Thr Ile Ile Thr Gln LeuAsn 485 490 495 Glu Val Pro Tyr Phe Gly Arg Gly Gly Asn Ile Met Ile SerAsn Ser 500 505 510 Leu Lys Ser His Gln Leu Glu Leu Arg Asn Tyr Thr ThrLys Lys Glu 515 520 525 Met Ser Ile Asn Asp Ile Tyr Val Arg Ala Thr SerGlu Glu Leu Tyr 530 535 540 Phe Tyr Ser Lys Lys Tyr Glu Lys Arg Val IlePhe Val Met Asn Asn 545 550 555 560 Met Phe Asn Tyr Ile Asn Gly Ser LysLeu Leu Arg Phe Leu Leu Glu 565 570 575 Val Ser Asn Ser Asp Phe Gln AsnIle Thr Pro Ile Thr Leu Gly Ser 580 585 590 Leu Asp Ser Tyr Asn His ValPro Ala Ile Ile Tyr Lys Asp Ile Ile 595 600 605 Ile Lys Pro Glu Thr TrpAsn Ile Arg Lys Ser Glu Ala Lys Thr Leu 610 615 620 Asp Ser Leu Lys AsnTrp Leu Thr Asn Asn Asn Val Pro Pro Phe Val 625 630 635 640 Arg Met LysTyr Thr Asp Gln Ile Ile Tyr Leu Asp Leu Ser Arg Thr 645 650 655 Ile AspLeu Thr Met Leu Phe Gln Ser Ile Lys Lys His Ser Phe Ile 660 665 670 GlnLeu Leu Asp Val His Ser Val Cys Thr Asn Asp Thr Glu Ile Leu 675 680 685Glu Leu Val Val Pro Phe Thr Arg Ser Asp Val Asn Ala His Gln Ile 690 695700 Tyr His Tyr Ala Gln Asn Ile Tyr Thr Leu Glu Asp Ser Gly Ser Lys 705710 715 720 Glu Lys Tyr Phe Tyr Ala Lys Ile Tyr Val Asn Lys Gln Arg GlnThr 725 730 735 Ser Phe Leu Gln Lys Glu Tyr Pro Leu Leu Leu Lys Tyr LeuLys Leu 740 745 750 Pro Glu Asn Leu Gln Trp Phe Tyr Ile Arg Tyr Lys AspAsp Gly Lys 755 760 765 Asp Ser Ile Arg Leu Arg Ile Arg Tyr Val Glu AspLys Gln Leu Val 770 775 780 Gln Leu Tyr Ser Arg Phe Ile Glu Trp Ala ThrLys Ala Arg Lys Asn 785 790 795 800 Ile Gln Ile Ser Gly Tyr Glu Ile SerGlu Tyr Ile Pro Glu Ser Ala 805 810 815 Arg Tyr Gly Gly Lys Lys Tyr SerSer Ile Ile His Ser Phe Phe Tyr 820 825 830 Tyr Asp Ser Ile Leu Asp LeuLeu Leu Gln Lys Lys Ala Glu Gln Thr 835 840 845 Ile Glu Val Arg Thr SerLeu Ser Ile Ile Arg Met Phe Leu Met Met 850 855 860 Lys Leu Ser Leu GlnAsp Gln Gln Lys Leu Ile Lys Asn Leu Phe Asp 865 870 875 880 Gly Lys HisLys Leu Lys Tyr Glu Lys Glu Tyr His Asn Ser Ile Ser 885 890 895 Leu LeuLeu Asp Asn Leu Cys Thr Lys Asn Gln Thr Asp Glu Ala Asp 900 905 910 IlePhe Cys Val Met Asn Met Lys Lys Ile Thr Glu Lys Ile Ser Ser 915 920 925Val Leu Lys Gln Lys Asp Leu Thr Thr Asp Trp Gln Arg Ile Leu Gly 930 935940 Ser Leu Ile His Met Arg Cys Asn Arg Val Tyr Gly Ile Asn Ser Glu 945950 955 960 Leu Glu Arg Lys Thr Met Phe Ile Val Asp Lys Val Ile Asn SerLys 965 970 975 Arg Tyr Thr Asp Met Phe Leu Glu Val Gly Asn Glu Thr Lys980 985 990 8 424 PRT Streptococcus mutans 8 Met Arg Gln Ser Lys Arg ValGlu Lys Ile Lys Asp Ile Leu Thr Glu 1 5 10 15 Gln Thr Tyr Leu Phe AspTyr Gln Glu Ile Leu Lys Lys Val Ser Gln 20 25 30 Ala Lys Gln Thr Asp PheTrp Asn Leu Leu Ser Leu Ser Ser Gly Ile 35 40 45 Thr Ser Leu Leu Ile PheTyr Gln Glu Tyr Glu Asn Leu Glu Gly Val 50 55 60 Asn Leu Lys Gln Gln LysGln Ser Leu Ile Gly Leu Ile Ser His Tyr 65 70 75 80 Ile Asn Gln Ile AlaGlu Lys Ser Ser Leu Phe Asp Gly Leu Ala Gly 85 90 95 Val Gly Phe Ala IleAsn Tyr Ile Ser Asn Asn Gly Lys Tyr Tyr Gln 100 105 110 Lys Leu Leu GluGln Ile Asp Asn Arg Leu Arg Gln Asn Ile Glu Arg 115 120 125 Asn Leu ValAsn Tyr Lys Asn Glu Glu Tyr Ala Asn Pro Met Asn Tyr 130 135 140 Asp ValVal Ser Gly Asn Ala Gly Val Ala Arg Tyr Leu Met Glu Arg 145 150 155 160Glu Ser Ser Glu Asp Trp Arg Ile Val Glu Met Ile Leu Glu Thr Phe 165 170175 Tyr Lys Ala Leu Glu Gln Gly Trp Arg Val Gln Ser Lys Tyr Gln Phe 180185 190 Leu Glu Ser Glu Lys Gln Tyr Tyr Leu Glu Gly Asn Ile Asn Phe Gly195 200 205 Leu Ala His Gly Ile Leu Gly Pro Ala Thr Ile Met Ala Leu TyrGln 210 215 220 Arg Arg Glu Pro Gln Asn Thr Arg Asn Ala Glu Lys Leu GlnGlu Thr 225 230 235 240 Tyr Arg Leu Ile Lys Arg Tyr Ala Gln Val Arg AspGlu Gly Leu Arg 245 250 255 Trp Pro Ile Arg Tyr Asp Leu Phe Arg Lys GluGly Ser Phe Ile Leu 260 265 270 Arg Asn Gly Trp Cys Tyr Gly Glu Asn GlyIle Tyr Asn Thr Leu Phe 275 280 285 Leu Met Gly Lys Val Leu Ser Asn GlnGlu Ile Cys Glu Thr Ala Gln 290 295 300 Lys Val Ile Pro Ser Ile Ile LysAsp Asp Tyr Glu Lys Met Glu Ser 305 310 315 320 Pro Thr Phe Cys His GlyPhe Ala Gly Lys Ala Asn Phe Phe Leu Leu 325 330 335 Gln Tyr Gln Arg ThrLys Glu Ser Ile Phe Leu Val Lys Ala Glu Glu 340 345 350 Glu Ile Asp LysIle Leu Ile Val Tyr Asn Ser Glu Asn Met Phe Gly 355 360 365 Phe Lys AspIle Glu Asp Asn Ile Asp Asn Thr Gly Glu Arg Leu Thr 370 375 380 Tyr TrpAsp Asn Phe Gly Leu Leu Ser Gly Thr Val Gly Val Leu Leu 385 390 395 400Val Leu Met Glu Tyr Cys Asn Ile Val Asn Ala Gly Lys Ile Ala Glu 405 410415 Trp Asn Lys Ile Phe Leu Leu Thr 420 9 188 PRT Streptococcus mutans 9Met Glu Glu Gln Asn Ile Glu Lys Lys Ile Leu Leu Cys Leu Thr Gly 1 5 1015 Ser Gly Ala Leu Leu Gly Ile Ala Glu Tyr Ile Thr Phe Leu Thr Val 20 2530 Arg Phe Lys His Val Arg Val Ile Val Ser Asp Asn Ala Ala Lys Met 35 4045 Leu Pro Val Ala Ala Ile Thr Gln Leu Cys Glu Lys Val Tyr Thr Asp 50 5560 Glu Val Ser Phe Thr Asp Lys Gln Lys Asn His Ile Ala Leu Thr Arg 65 7075 80 Trp Ala Asp Ile Thr Val Val Leu Pro Ala Thr Ala Asn Ile Ile Gly 8590 95 Lys Val Ala Asn Gly Ile Ala Asp Asn Phe Met Thr Thr Thr Leu Leu100 105 110 Ser Ser Ser Lys Pro Val Leu Ile Tyr Pro Cys Met Asn Asn IleMet 115 120 125 Trp Glu Asn Pro Val Val Gln Lys Asn Val Glu Val Leu SerGly Thr 130 135 140 Gln Tyr Lys Val Ile Val Gly Gln Glu Ser Glu Ser PheGlu Leu Ala 145 150 155 160 Ser Gly Lys Met Lys Lys Asn Ile Ala Ile ProSer Leu Asp Glu Leu 165 170 175 Gln Arg Val Val Leu Glu Asn Leu Gln GluGlu Arg 180 185 10 447 PRT Streptococcus mutans 10 Met Lys Lys Lys GlyLeu Leu Val Ile Ile Phe Leu Thr Phe Phe Phe 1 5 10 15 Phe Tyr Pro LysAla Lys Ala Ala Glu Tyr Thr Ile Ile Ser Asn Asn 20 25 30 Ser Glu Gln ThrVal Asn Asp Leu Asn Asn Leu Gly Val Thr Val Asn 35 40 45 Ser His Ile AlaGlu Ile Gly Tyr Ile Glu Ala Gln Gly Asp Val Asn 50 55 60 Ile Asp Gln IleLys Lys Leu Ser Asn Ile Gln Ser Ile Gln Asn Met 65 70 75 80 Ala Asp ThrSer Gln Asn Ile Thr Thr Arg Val Pro Ser Thr Tyr Ile 85 90 95 Asn Gln ThrIle Gln Leu Pro Gln Leu Phe Ser Tyr Gln Trp Asp Met 100 105 110 Gln LysIle Thr Asn Asn Gly Val Ser Tyr Ser Leu Asn Lys Glu Asn 115 120 125 ArgLys Asn Val Thr Val Ala Leu Val Asp Ser Gly Ile Asp Val Asp 130 135 140His Asn Ala Phe Thr Gly Met Ile Asp Ser Arg Ser Lys Asn Phe Val 145 150155 160 Pro Ala Gly Gly Tyr Asp Asn Ser Glu Ser Ser Glu Thr Gly Asn Ile165 170 175 Asn Asp Ile Asp Asp Lys Lys Gly His Gly Thr Ala Val Ala GlyGln 180 185 190 Ile Ala Ala Asn Gly Gln Ile Phe Gly Val Ser Pro Gly ThrAsn Leu 195 200 205 Leu Ile Tyr Arg Val Phe Gly Lys Ser Lys Ser Lys GluCys Trp Ile 210 215 220 Leu Lys Ala Ile Ile Asp Ala Thr Asn Asn Gly AlaAsn Val Ile Asn 225 230 235 240 Leu Ser Leu Gly Gln Tyr Ile Lys Ile ProAsn Gly Asp Ile Trp Glu 245 250 255 Ser Ala Glu Ala Leu Gly Tyr Lys PheAla Ile Asp Tyr Ala Thr Arg 260 265 270 His Asn Val Ile Val Val Ala AlaThr Gly Asn Asp Gly Leu Ser Asp 275 280 285 Asp Asn Gly Glu Val Lys ThrTyr Tyr Asn Ser Gln His Ser Gly Gln 290 295 300 Asp Met Ser Gln Asn AspThr Val Glu Asp Tyr Pro Ser Val Leu Pro 305 310 315 320 Asn Ala Ile AlaVal Gly Ser Ser Asp Asn Asn Asn Gln Arg Ser Ser 325 330 335 Phe Ser AsnTyr Tyr Asn Gln Tyr Gln Asp Asn Phe Ile Leu Ala Pro 340 345 350 Gly GlyGly Thr Thr Leu Leu Asp Gln Tyr Gly Gln Glu Glu Trp Tyr 355 360 365 AsnGln Lys Leu Phe Met Lys Glu Gln Val Leu Ser Thr Ser Asn Asn 370 375 380Gly Asn Tyr Asp Tyr Ala Asp Gly Thr Ser Ile Ser Thr Gly Lys Val 385 390395 400 Ser Gly Glu Leu Ala Glu Ile Ile Ser Asn Tyr His Leu Gln Gly Asp405 410 415 Ser Ser Lys Ala Arg Ser Ile Leu Leu Asn Gln Val Asn Tyr ThrSer 420 425 430 Asp Gly Tyr Lys Glu Ile Ser Thr Tyr Lys Ala Leu Arg GlyTyr 435 440 445 11 541 PRT Streptococcus mutans 11 Met Lys Trp Leu GluVal Leu Gln Ile Ser Lys Lys Glu Lys Ile Leu 1 5 10 15 Tyr Leu Ile GlyCys Ile Phe Ser Ile Met Thr Gly Leu Ile Thr Leu 20 25 30 Arg Ile Thr TyrLeu Leu Lys Asn Leu Val Asp Ser Lys Ser Ser Phe 35 40 45 Asn Asn Leu PheLeu Phe Leu Val Leu Gly Leu Val Leu Phe Ile Ile 50 55 60 Asp Ala Gly SerGln Tyr Leu Ile Ser Leu Ile Gly Asn Gln Val Val 65 70 75 80 Phe Asn SerArg Asn Asn Ile Trp Lys Lys Ile Ser Asp Trp Thr Asp 85 90 95 Ser Lys AspAsp Ser Ser Glu Met Ala Gly His Leu Ile Asn Asp Ser 100 105 110 Glu LeuIle Glu Asn Phe Ile Ile Ser Thr Ile Pro Gln Ser Ile Asn 115 120 125 SerVal Ile Val Gly Ser Gly Ser Leu Val Met Leu Phe Val Ile Asn 130 135 140Ser Lys Met Ser Leu Glu Val Ile Gly Ile Cys Leu Leu Leu Leu Phe 145 150155 160 Ile Met Gln Pro Phe Ser Arg Ile Leu Ser Lys Ile Ser Lys Arg Ile165 170 175 Gln Glu Asp Lys Ala Glu Leu Ile Asn Ile Ala Ser Gln Leu ArgGly 180 185 190 Gln Val Lys Thr Ile Lys Ser Tyr Asn Ala Gln Asp Tyr AlaPhe Gln 195 200 205 Lys Phe Asp Glu Gln Asn Arg Gln Leu Phe Gln Asp IleLeu Asn Arg 210 215 220 Ile Lys Ile Phe Ser Ile Tyr Ser Pro Phe Leu AsnIle Leu Ile Leu 225 230 235 240 Phe Met Ile Ile Ile Val Val Trp Leu GlyAsn Thr Glu Val Arg Ser 245 250 255 Gly Asn Leu Thr Val Gly Ser Ala ThrIle Phe Val Val Tyr Met Thr 260 265 270 Gln Leu Ile Asn Pro Ile Met GlnLeu Ser Gln Leu Val Ala His Met 275 280 285 Gly Met Leu Asn Gly Gly ValGlu Arg Leu Leu Glu Tyr Asn Gln Ala 290 295 300 Ile Pro Glu Lys Asn GlyIle Lys Lys Ile Asp Glu Ile Ile Asn Ile 305 310 315 320 Ala Phe Asp AsnVal Ser Phe Ala Tyr Asp Asn Gln Glu Asn Ile Ile 325 330 335 Glu Asn ValAsn Leu Thr Phe Gln Lys Gly Thr Tyr Ile Ser Ile Val 340 345 350 Gly GluSer Gly Val Gly Lys Ser Thr Leu Leu Asp Leu Leu Glu His 355 360 365 AsnTyr Val Pro Ser Lys Gly Arg Ile Leu Ile Asn Gly Ile Asp Leu 370 375 380Glu Glu Leu Asn Ile Lys Thr Leu Arg Asn Lys Ile Ser Tyr Val Ser 385 390395 400 Gln Glu Pro Thr Ile Leu Ser Gly Thr Ile Arg Glu Leu Leu Asp Phe405 410 415 Asn Gln Gln Gln His Thr Glu Thr Ser Leu Trp Asn Val Leu AspThr 420 425 430 Val Glu Leu Ser Glu Leu Ile Arg Asn Leu Pro Ala Lys LeuAsp Ser 435 440 445 Lys Val Asp Glu Tyr Gly Gly Asn Leu Ser Gly Gly GlnMet Gln Arg 450 455 460 Ile Ser Leu Ala Arg Gly Leu Leu Lys Ala Gly AspVal Leu Leu Leu 465 470 475 480 Asp Glu Ser Phe Ala Asn Ile Asp Glu GluThr Cys Leu Lys Ile Lys 485 490 495 Leu Lys Ile Ala Ala Tyr Ala Glu SerHis Lys Gln Ile Val Ile Glu 500 505 510 Val Ile His Asn Leu Asn Arg IleThr Pro Ser Ser Ile Val Tyr Arg 515 520 525 Leu Ala Asp Lys Lys Leu GluIle Leu Arg Ser Gly Phe 530 535 540 12 233 PRT Streptococcus mutans 12Met Asp Tyr Met Leu Glu Thr Lys Asn Leu Thr Lys Gln Phe Gly Lys 1 5 1015 Gln Thr Ala Val Asn Gln Leu Asn Leu Lys Val Glu Arg His Ser Ile 20 2530 Tyr Gly Leu Leu Gly Pro Asn Gly Ser Gly Lys Ser Thr Thr Leu Lys 35 4045 Met Ile Thr Gly Met Leu Arg Lys Thr Ser Gly His Ile Leu Ile Asp 50 5560 Gly His Asp Trp Ser Arg Lys Asp Leu Glu Asn Ile Gly Ala Leu Ile 65 7075 80 Glu Ser Pro Pro Leu Tyr Glu Asn Leu Thr Ala Arg Glu Asn Leu Lys 8590 95 Val Arg Thr Leu Met Leu Gly Leu Pro Asp Ser Arg Ile Asp Glu Val100 105 110 Leu Lys Ile Val Asp Leu Thr Asn Thr Gly Lys Lys Arg Ala GlyGln 115 120 125 Phe Ser Met Gly Met Lys Gln Arg Leu Gly Ile Ala Ile AlaLeu Leu 130 135 140 Asn Ser Pro Gln Leu Leu Ile Leu Asp Glu Pro Thr AsnGly Leu Asp 145 150 155 160 Pro Ile Gly Ile Gln Glu Leu Arg Asn Leu IleArg Ser Phe Pro Thr 165 170 175 Gln Gly Ile Thr Val Ile Ile Ser Ser HisIle Leu Ser Glu Ile Gln 180 185 190 Met Thr Ala Asp His Ile Gly Ile IleAla Asn Gly Val Leu Gly Tyr 195 200 205 Gln Asp Arg Ile His Gln Asp GluAsp Leu Glu Lys Leu Phe Thr Asp 210 215 220 Val Val Met Arg Tyr Arg GlyGly Glu 225 230 13 251 PRT Streptococcus mutans 13 Met Leu Gly Met PheGln Ala Glu Arg Leu Lys Leu Lys Arg Ser Met 1 5 10 15 Ala Lys Lys LeuLeu Val Phe Ala Pro Ile Ile Ala Ile Leu Tyr Gly 20 25 30 Phe Ile Ala ProVal Gly Tyr Leu Val Asn Asn Ala Tyr Asn Trp Trp 35 40 45 Tyr Val Met IlePhe Pro Gly Leu Leu Thr Leu Phe Ala Ala Leu Ile 50 55 60 Asn Thr Tyr GluGlu Lys Lys Leu His Tyr Arg Ala Val Phe Pro Leu 65 70 75 80 Pro Ile SerLeu Arg Lys Phe Trp Phe Glu Lys Ile Phe Ile Thr Val 85 90 95 Tyr Tyr LeuAsn Phe Ser Asn Gly Val Leu Trp Ile Ile Thr Val Leu 100 105 110 Leu AsnThr Phe Ile Leu Pro Asn Tyr Gly Lys Asp Tyr Thr Tyr Thr 115 120 125 ValGly Glu Leu Ala Leu Ala Ser Leu Val Ile Ile Val Thr Thr Leu 130 135 140Trp Gln Ile Pro Phe Cys Leu Trp Leu Thr Lys Arg Ile Gly Phe Thr 145 150155 160 Ile Thr Leu Ile Ile Asn Leu Met Ser Asn Phe Ile Leu Gly Val Val165 170 175 Phe Ala Thr Thr Ser Cys Trp Trp Leu Cys Pro Tyr Ser Trp GlyIle 180 185 190 Arg Leu Met Val Pro Ile Leu Lys Ile Leu Pro Ser Gly LeuLys Ala 195 200 205 Gly Ile Ala Gly Ala Pro Ser Leu Pro Thr Ser Phe TrpSer Ile Val 210 215 220 Ile Ser Leu Cys Leu Ala Val Ile Leu Phe Val SerLeu Thr Val Leu 225 230 235 240 Ser Ala Ser Trp Phe Glu Lys Gln Glu ValLys 245 250 14 246 PRT Streptococcus mutans 14 Met Ile Asp Leu Leu LysAla Glu Asn Val Lys Tyr Arg His Thr Phe 1 5 10 15 Leu Pro Trp Leu HisLeu Ile Leu Pro Val Thr Thr Ala Ile Val Val 20 25 30 Ile Val Tyr Gly LeuMet Thr Pro Thr His Ser Trp Ala Asp Ile Thr 35 40 45 Gly Gly Tyr Leu GluLeu Leu Gly Ile Ser Phe Pro Ile Val Ile Ala 50 55 60 Val Ile Cys Gly LysSer Val Gly Leu Glu Val Glu Ala Gly Gln Phe 65 70 75 80 Gln Val Met LeuAla Ile Lys Gln Arg Asn Leu Ile Phe Cys Ile Lys 85 90 95 Leu Leu Asn LeuLeu Ile Leu Glu Leu Phe Ser Thr Leu Leu Ala Ile 100 105 110 Gly Ile TyrGly Leu Ile Tyr Gln Leu Ser Asn Lys His Leu Ile Phe 115 120 125 Tyr GlyTyr Ala Val Ile Leu Leu Thr Ala Ser Met Leu Ile Leu Tyr 130 135 140 LeuIle His Leu Val Val Val Phe Leu Phe Gly Asn Ser Ala Asn Ile 145 150 155160 Gly Leu Gly Ile Ala Glu Ser Leu Leu Ser Ala Leu Leu Leu Thr Gly 165170 175 Leu Gly Asp Gly Ile Trp Gln Phe Ile Pro Cys Ala Trp Gly Thr Arg180 185 190 Leu Met Gly Thr Leu Ile Asn Leu Trp Tyr Tyr Ser Gly His SerLeu 195 200 205 Phe Phe Lys Gln Gln Leu Leu Ile Trp Leu Glu Val Ala ValPro Leu 210 215 220 Thr Leu Met Ala Leu Ile Leu Ser Ile Ile Trp Phe AspArg Trp Gln 225 230 235 240 Gly Arg Ser Ser Asp Glu 245 15 246 PRTStreptococcus mutans 15 Met Thr Tyr Ile Gly Val Ser His Leu Lys Lys ValTyr Lys Thr Gln 1 5 10 15 Glu Gly Leu Thr Asn Glu Ala Leu Lys Asp IleThr Phe Ser Val Gln 20 25 30 Glu Gly Glu Phe Ile Ala Ile Met Gly Glu SerGly Ser Gly Lys Ser 35 40 45 Thr Leu Leu Asn Ile Leu Ala Cys Met Asp TyrPro Ser Ser Gly His 50 55 60 Ile Ile Phe Asn Asn Tyr Gln Leu Glu Lys ValLys Asp Glu Glu Ala 65 70 75 80 Ala Val Phe Arg Ser Arg His Ile Gly PheIle Phe Gln Asn Phe Asn 85 90 95 Leu Leu Asn Ile Phe Asn Asn Lys Asp AsnLeu Leu Ile Pro Val Ile 100 105 110 Ile Ser Gly Ser Lys Val Asn Ser TyrGlu Lys Arg Leu Arg Asp Leu 115 120 125 Ala Ala Val Val Gly Ile Glu SerLeu Leu Ser Lys Tyr Pro Tyr Glu 130 135 140 Leu Ser Gly Gly Gln Gln GlnArg Leu Ala Ile Ala Arg Ala Leu Ile 145 150 155 160 Met Asn Pro Asp LeuIle Leu Ala Asp Glu Pro Thr Gly Gln Leu Asp 165 170 175 Ser Lys Thr SerGln Arg Ile Leu Asn Leu Leu Ser Asn Ile Asn Ala 180 185 190 Lys Arg LysThr Ile Leu Met Val Thr His Ser Pro Lys Ala Ala Ser 195 200 205 Tyr AlaAsn Arg Val Leu Phe Ile Lys Asp Gly Val Ile Phe Asn Gln 210 215 220 LeuVal Arg Gly Cys Lys Ser Arg Glu Gly Phe Leu Asp Gln Ile Ile 225 230 235240 Met Ala Gln Ala Ser Leu 245 16 640 PRT Streptococcus mutans 16 MetPhe Leu Pro Lys Ile Ser Phe His Asn Leu Ile Val Asn Lys Ser 1 5 10 15Leu Thr Leu Pro Tyr Phe Ala Ile Met Thr Ile Phe Ser Gly Phe Asn 20 25 30Tyr Val Leu Ile Asn Phe Leu Thr Asn Pro Ser Phe Tyr Asn Ile Pro 35 40 45Thr Ala Arg Ile Leu Ile Asp Ile Leu Ile Phe Gly Phe Ile Leu Ile 50 55 60Ser Leu Leu Met Leu Leu Tyr Gly Arg Tyr Ala Asn Arg Phe Ile Ser 65 70 7580 Asp Glu Arg Asn Ser Asn Met Gly Ile Phe Leu Met Leu Gly Met Gly 85 9095 Lys Lys Gln Leu Leu Lys Ile Ile Tyr Leu Glu Lys Leu Tyr Leu Phe 100105 110 Thr Gly Thr Phe Phe Gly Gly Leu Ile Phe Gly Phe Val Tyr Ser Lys115 120 125 Ile Phe Phe Leu Phe Ile Arg Asn Leu Ile Val Ile Gly Asp ValArg 130 135 140 Glu Gln Tyr Ser Leu Thr Ala Ile Ser Trp Leu Leu Ile LeuThr Phe 145 150 155 160 Phe Ile Tyr Phe Ile Ile Tyr Leu Ser Glu Tyr ArgLeu Leu Lys Arg 165 170 175 Gln Ser Ile Thr Val Ile Phe Asn Ser Lys AlaLys Arg Asp Asn Pro 180 185 190 Arg Lys Thr Ser Val Phe Val Gly Leu PheGly Leu Phe Ala Leu Leu 195 200 205 Met Gly Tyr His Phe Ala Leu Thr SerPro Asn Val Thr Thr Ser Phe 210 215 220 Ser Arg Phe Ile Tyr Ala Ala CysLeu Val Thr Leu Gly Ile Phe Cys 225 230 235 240 Thr Phe Ser Ser Gly ValIle Met Leu Leu Thr Val Ile Lys Lys Arg 245 250 255 Arg Ala Ile Tyr TyrAsn Gln Arg Arg Phe Val Val Ile Ala Ser Leu 260 265 270 Phe His Arg IleArg Ser Asn Ala Leu Ser Leu Ala Thr Ile Cys Ile 275 280 285 Phe Ser ThrAla Thr Leu Val Ser Leu Ser Val Leu Ala Ser Leu Tyr 290 295 300 Leu AlaLys Asp Asn Met Val Arg Leu Ser Ser Pro Arg Asp Val Thr 305 310 315 320Val Leu Ser Thr Thr Asp Ile Glu Pro Asn Leu Met Asp Ile Ala Thr 325 330335 Lys Asn His Val Thr Leu Thr Asn Arg Gln Asn Leu Lys Val Ser Gln 340345 350 Ser Val Tyr Gly Asn Ile Lys Gly Ser His Leu Ser Val Asp Pro Asn355 360 365 Gly Gly Met Ala Asn Asp Tyr Gln Ile Thr Val Ile Ser Leu AspSer 370 375 380 Phe Asn Ala Ser Asn Asn Thr His Tyr Arg Leu Lys Asn HisGlu Ile 385 390 395 400 Leu Thr Tyr Val Ser Asn Gly Ala Ala Ala Pro SerSer Tyr Thr Thr 405 410 415 Asn Gly Val Lys Leu Thr Asn Val Lys Gln IleLys Arg Ile Asn Phe 420 425 430 Ile Phe Ser Pro Leu Arg Ser Met Gln ProAsn Phe Phe Ile Ile Thr 435 440 445 Asp Asn Arg Glu Ile Ile Gln Thr IleLeu Lys Glu Glu Leu Thr Trp 450 455 460 Gly Thr Met Ala Gly Tyr His ValLys Gly Lys Lys Met Asn Gln Lys 465 470 475 480 Asp Phe Tyr Asp Glu LeuGlu Thr Thr Asn Phe Arg Gln Phe Ser Ala 485 490 495 Asn Val Val Ser IleArg Gln Val Lys Ser Met Phe Asn Ala Leu Phe 500 505 510 Gly Gly Leu LeuPhe Val Gly Ile Ile Phe Gly Thr Ile Phe Ala Ile 515 520 525 Leu Thr AlaIle Thr Ile Tyr Tyr Gln Gln Leu Ser Glu Gly Ile Arg 530 535 540 Asp ArgAsp Asp Tyr Lys Ala Met Ile Lys Leu Gly Met Thr Asn Lys 545 550 555 560Thr Ile Gln Asp Ser Ile Lys Val Gln Ile Asn Phe Val Phe Ile Leu 565 570575 Pro Ile Ala Phe Ala Leu Leu Asn Leu Ile Phe Ala Leu Pro Ile Leu 580585 590 Tyr Lys Ile Met Thr Thr Phe Gly Phe Asn Asp Ala Gly Leu Phe Leu595 600 605 Arg Ala Val Gly Thr Cys Leu Ile Val Tyr Leu Phe Phe Tyr TrpPhe 610 615 620 Ile Cys His Cys Thr Ser Lys Leu Tyr Tyr Arg Leu Ile SerLys Lys 625 630 635 640 17 118 PRT Streptococcus mutans 17 Met Arg IleVal Ser Ser Leu Val Ser Leu Leu Leu Thr Ile Phe Trp 1 5 10 15 Ile PheAla Ile Ala Phe Ile Pro Ile Gly Asp Gln Asn Ser Phe Asn 20 25 30 Lys ProGlu Met Trp Phe Phe Val Phe Phe Ala Ile Ile Ile Tyr Ser 35 40 45 Ile ValIle Ile Ser Asp Tyr Tyr Leu Lys Ser Phe Asn Leu Leu Lys 50 55 60 Val TyrGln Ile Leu Val Leu Phe Ile Ser Ile Leu Cys Ala Leu Cys 65 70 75 80 GlyLeu Ser Leu Thr Ala Leu Gly Leu Lys Val Phe Thr Leu Ala Ile 85 90 95 GlyIle Val Ser Leu Val Asn Thr Ile Ile Tyr Phe Phe Phe Ala Asn 100 105 110Lys Lys Asp Asn Val Glu 115 18 63 PRT Streptococcus mutans 18 Met SerAsn Thr Gln Leu Leu Glu Val Leu Gly Thr Glu Thr Phe Asp 1 5 10 15 ValGln Glu Asp Leu Phe Ala Phe Asp Thr Thr Asp Thr Thr Ile Val 20 25 30 AlaSer Asn Asp Asp Pro Asp Thr Arg Phe Lys Ser Leu Ser Leu Cys 35 40 45 ThrPro Gly Cys Ala Arg Thr Gly Ser Phe Asn Ser Tyr Cys Cys 50 55 60 19 51PRT Streptococcus epidermis 19 Met Glu Ala Val Lys Glu Asn Asp Leu PheAsn Leu Asp Val Lys Val 1 5 10 15 Asn Ala Lys Glu Ser Asn Asp Ser GlyAla Glu Pro Arg Ile Ala Ser 20 25 30 Lys Phe Ile Cys Thr Pro Gly Cys AlaLys Thr Gly Ser Phe Asn Ser 35 40 45 Tyr Cys Cys 50

1. A method of making mutacin I protein comprising culturing amicroorganism transformed with DNA (SEQ ID No: 1) encoding for mutacin Iprotein and recovering mutacin I protein.
 2. A method according to claim1, wherein said culturing step comprises disposing the microorganisms ona membrane.
 3. A method according to claim 2 wherein said culturing stepcomprises contacting the membrane having the microorganism thereon witha growth media.
 4. A method according to claim 3, further comprising thestep of freezing the growth media having the microorganism thereon andthen thawing the growth media.
 5. A method according to claim 4, furthercomprising the step of separating a liquid fraction of the growth mediafrom a solid fraction of the growth media.
 6. A method according toclaim 5, wherein said recovery step comprises the step ofchromatographically separating the mutacin I from the liquid fraction.7. A process for the production of mutacin I having part or all of theprimary structural conformation and biological activity of bacterialmutacin I, said process comprising: growing, under suitable conditions,prokaryotic or eukaryotic host cells transformed or transfected with aDNA sequence according to claim 1 in a manner allowing expression ofsaid mutacin I product; and isolating the mutacin I product of theexpression of said DNA sequence.
 8. A mutacin I protein product of theexpression in a prokaryotic or eukaryotic host cell of DNA according toclaim
 7. 9. A method of treating or preventing an infection in asubject, said method comprising administering to said subject aneffective amount of a purified and isolated peptide having the aminoacid sequence as set forth in SEQ ID No: 2, or a pharmaceuticallyacceptable salt, amide, ester, or prodrug thereof.
 10. A methodaccording to claim 9, wherein the peptide is administered orally.
 11. Amethod according to claim 9, wherein the peptide is administeredtopically.
 12. A method according to claim 9, wherein the peptide isapplied to a surface of a medical device.
 13. A method according toclaim 12, wherein the medical device is a catheter.
 14. A methodaccording to claim 12, further including the step of coating the medicaldevice with the peptide prior to contacting the subject therewith.